Inhibition of fatty acid synthesis by iodo-nitrobenzamide compounds and methods of treatment thereof

ABSTRACT

The present invention relates to a method of treating a fatty acid synthesis related disease comprising administering to a patient in need thereof an effective amount of a PARP inhibitor or metabolite thereof to inhibit fatty acid synthesis, wherein the fatty acid synthesis related disease is obesity, diabetes, or cardiovascular disease. The present invention also relates to a method of treating a cancer in a subject comprising: (i) identifying a level of fatty acid in a sample from the subject, and (ii) administering an effective amount of a PARP inhibitor or metabolite thereof to inhibit fatty acid synthesis in the subject, wherein the administration is based on the level of fatty acid, thereby treating the cancer in the subject. The present invention further relates to a method of treating Her-2 related cancers by administering to a patient in need thereof an effective amount of a PARP inhibitor or metabolite thereof to inhibit fatty acid synthesis.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/842,479, filed Sep. 5, 2006, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Fatty acid synthase (FAS) is an enzyme required to convert carbohydratesto fatty acids. In normal humans, the fatty acid synthetic pathway isdown-regulated because of sufficiently high levels of dietary fat.However, in a wide variety of human malignancies and their precursorlesions, such as, prostate cancer, ovarian cancer, and breast cancer,the tissues express high levels of fatty acid synthase resulting in highlevels of fatty acids. An up-regulation of FAS in most human cancersleads to the notion that FAS plays a role in the development,maintenance, and/or enhancement of the malignant cancer phenotype andthat FAS can be a target for anticancer drug development.

About 1.2 billion people in the world are overweight and at least 300million of them are obese. In the United States, more than 97 millionadults—that's more than half—are overweight and almost one in fiveadults is obese. Reduction of the fatty acid synthesis in the obese canbe an effective treatment for obesity.

Treatment of cancer cells in vitro with cerulenin, a covalentinactivator of the β-ketoacyl synthase reaction on FAS, led to celldeath by means of apoptosis, demonstrating that cancer cells with highlyactive fatty acid synthesis require a functional pathway (Pizer et al.(1996) Cancer Res. 56, 2745-2747). Cerulenin, however, has limited invivo activity. FAS has been shown to be one of the genes regulated byHer-2/neu at the level of transcription, translation, and biosyntheticactivity (Menedez et al. (2005) Drug New Perspect, 18(6), July/August).This Her-2/neu-induced up-regulation of breast-cancer associated FAS isinhibitable by anti-Her-2/neu antibodies such as trastuzumab. Studieswith C75, an inhibitor of fatty acid synthesis, have shown anti-tumoractivity with inhibition of fatty acid synthesis in tumor tissue(Kuhajda et al. (2000) Proc. Natl. Acad. Sci. vol. 97, no. 7,3450-3454). FAS inhibitors have also been shown to activateweight-reducing pathways (Loftus, T. M. et al. (2000) Science 288,2379-2381).

As fatty acid synthesis plays a role in cancer and weight gain pathways,there continues to be a need to develop effective fatty acid synthesisinhibitors.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method of treating a fatty acidsynthesis and metabolism related disease comprising administering to apatient in need thereof an effective amount of a PARP inhibitor ormetabolite thereof to inhibit fatty acid synthesis, wherein the fattyacid synthesis related disease is obesity, diabetes, or cardiovasculardisease.

Another aspect of the present invention relates to a method of treatinga cancer in a subject comprising: (i) identifying a level of fatty acidin a sample from the subject, and (ii) administering an effective amountof a PARP inhibitor or metabolite thereof to inhibit fatty acidsynthesis wherein the administration is based on the level of fattyacid, thereby treating the cancer in the subject.

In some embodiments of the present invention, the fatty acid is a mediumchain fatty acid or a long chain fatty acid. The medium chain fattyacids include C:6-C:12. The long chain fatty acids include a chainlength of greater than 12 carbons. In some embodiments, the inhibitionof the fatty acid synthesis comprises inhibiting at least one enzyme ofa glucose pathway. In some embodiments, the inhibition of the fatty acidsynthesis comprises inhibiting at least one enzyme of a fatty acidbiosynthetic pathway. In some embodiments, the inhibition of the fattyacid synthesis comprises inhibiting at least one enzyme selected fromthe group consisting of acetyl Co-A, malonyl Co-A, acetyl Co-Acarboxylase, and fatty acid synthase. In some embodiments, the fattyacid synthase comprises acyl carrier protein, acetyl transferase,malonyl transferase, 3-keto-acyl-ACP synthase, 3-ketoacyl-ACP reductase,3-hydroxy-acyl-ACP dehydratase, and enoyl-ACP reductase. In someembodiments, the inhibition of the fatty acid synthesis comprisesinhibiting at least one enzyme of a fatty acid synthase. In someembodiments, the inhibition of the fatty acid synthesis comprisesinhibiting synthesis of an acetyl-CoA from a glucose. In someembodiments, the inhibition of the fatty acid synthesis comprisesinhibiting the fatty acid synthesis from an acetyl-CoA.

In some embodiments of the aforementioned aspect of the presentinvention, the long chain fatty acid is C:14-C:30. In some embodiments,the long chain fatty acid is C:14, C:16, C:18, C:18-1, C:20, C:22, orC:24. In some embodiments, the inhibition is determined by analyzing ametabolite or a molecular flux of a glucose pathway or a fatty acidbiosynthetic pathway. In some embodiments, the metabolite is selectedfrom the group consisting of glucose, glycogen, lactate, CO₂, fattyacid, acetyl Co-A, RNA ribose and DNA deoxyribose. In some embodiments,the metabolite is chemically derivatized for the analysis. In someembodiments, the analysis comprises mass spectrometry. In someembodiments, the mass spectrometry is mass isotopomer distributionanalysis. In some embodiments, the level of fatty acid is up-regulated.

In some embodiments of the aforementioned aspect of the presentinvention, the PARP inhibitor or metabolite thereof is a compound offormula I, its pharmaceutically acceptable salts or prodrugs thereof:

wherein, R₁, R₂, R₃, R₄, and R₅ are independently selected from thegroup consisting of hydrogen, hydroxy, optionally substituted amine,carboxyl, ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆)alkyl, optionally substituted (C₁-C₆) alkoxy, optionally substituted(C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇) heterocyclic,optionally substituted aryl and a sulfur-containing moiety. In someembodiments, the sulfur containing moiety is —SR₆, wherein R₆ isselected from the group consisting of hydrogen, C₁-C₆ acyl, optionallysubstituted (C₁-C₆) alkyl, optionally substituted (C₃-C₇) cycloalkyl,optionally substituted (C₃-C₇) heterocyclic and optionally substitutedaryl.

In some preferred embodiments of the aforementioned aspect of thepresent invention, the PARP inhibitor or metabolite thereof is acompound of formula II, its pharmaceutically acceptable salts orprodrugs thereof:

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid.

In some preferred embodiments of the aforementioned aspect of thepresent invention, the PARP inhibitor is a compound of formula III, itspharmaceutically acceptable salts, metabolites or prodrugs thereof:

In some embodiments of the aforementioned aspect of the presentinvention, the cancer is selected from the group consisting of colonadenocarcinoma, esophagus adenocarcinoma, liver hepatocellularcarcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet celltumor, rectum adenocarcinoma, gastrointestinal stromal tumor, stomachadenocarcinoma, adrenal cortical carcinoma, follicular carcinoma,papillary carcinoma, breast cancer, ductal carcinoma, lobular carcinoma,intraductal carcinoma, mucinous carcinoma, phyllodes tumor, ovarianadenocarcinoma, endometrium adenocarcinoma, granulose cell tumor,mucinous cystadenocarcinoma, cervix adenocarcinoma, vulva squamous cellcarcinoma, basal cell carcinoma, prostate adenocarcinoma, giant celltumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma,kidney carcinoma, urinary bladder carcinoma, Wilm's tumor, and lymphoma.

In some embodiments of the aforementioned aspect of the presentinvention, the treatment is selected from the group consisting of oraladministration, transmucosal administration, buccal administration,nasal administration, inhalation, parental administration, intravenous,subcutaneous, intramuscular, sublingual, transdermal administration, andrectal administration.

In some embodiments of the aforementioned aspect of the presentinvention, the sample from the subject is selected from the groupconsisting of tumor tissue, hair, blood, cell, tissue, organ, braintissue, blood, serum, sputum, saliva, plasma, nipple aspirant, synovialfluid, cerebrospinal fluid, sweat, urine, fecal matter, pancreaticfluid, trabecular fluid, cerebrospinal fluid, tears, bronchial lavage,swabbing, bronchial aspirant, semen, prostatic fluid, precervicularfluid, vaginal fluids, and pre-ejaculate.

Yet another aspect of the present invention relates to a method ofmonitoring a therapeutic effectiveness of a PARP inhibitor or metabolitethereof in a treatment of a disease comprising: (i) administering aneffective amount of a PARP inhibitor or metabolite thereof to a patientto inhibit fatty acid synthesis; (ii) comparing a first and a secondlevel of fatty acid in a first and second sample respectively, from thepatient wherein the first level and the first sample are obtained priorto administration of the PARP inhibitor or metabolite thereof and thesecond level and the second sample are obtained after administration ofthe PARP inhibitor or metabolite thereof; and (iii) determining atherapeutic effectiveness of the PARP inhibitor or metabolite thereof ina treatment of a disease in the patient based on the comparison.

In some embodiments of the aforementioned aspect of the presentinvention, the inhibition of the fatty acid synthesis comprisesinhibiting at least one enzyme of a glucose pathway or a fatty acidbiosynthetic pathway. In some embodiments, when the second level offatty acid in the second sample is lower than the first level of fattyacid in the first sample then the PARP inhibitor or metabolite thereofis therapeutically effective. In some embodiments, when the second levelof fatty acid in the second sample is higher than the first level offatty acid in the first sample then the PARP inhibitor or metabolitethereof is therapeutically ineffective. In some embodiments, the firstlevel and the second level of fatty acid is determined by assaytechniques. In some embodiments, the first level and the second level offatty acid is determined by mass spectrometry. In some embodiments, themass spectrometry is mass isotopomer distribution analysis.

In some embodiments of the aforementioned aspect of the presentinvention, the PARP inhibitor or metabolite thereof is a compound offormula I, its pharmaceutically acceptable salts or prodrugs thereof:

wherein, R₁, R₂, R₃, R₄, and R₅ are independently selected from thegroup consisting of hydrogen, hydroxy, optionally substituted amine,carboxyl, ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆)alkyl, optionally substituted (C₁-C₆) alkoxy, optionally substituted(C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇) heterocyclic,optionally substituted aryl and a sulfur-containing moiety. In someembodiments, the sulfur containing moiety is —SR₆, wherein R₆ isselected from the group consisting of hydrogen, C₁-C₆ acyl, optionallysubstituted (C₁-C₆) alkyl, optionally substituted (C₃-C₇) cycloalkyl,optionally substituted (C₃-C₇) heterocyclic and optionally substitutedaryl.

In some embodiments of the aforementioned aspect of the presentinvention, the PARP inhibitor or metabolite thereof is a compound offormula II, its pharmaceutically acceptable salts or prodrugs thereof:

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid.

In some embodiments of the aforementioned aspect of the presentinvention, the PARP inhibitor is a compound of formula III, itspharmaceutically acceptable salts or prodrugs thereof:

In some embodiments of the aforementioned aspect of the presentinvention, the disease is cancer, cardiovascular, diabetes and obesity.In some embodiments, the administration is selected from the groupconsisting of oral administration, transmucosal administration, buccaladministration, nasal administration, inhalation, parentaladministration, intravenous, subcutaneous, intramuscular, sublingual,transdermal administration, and rectal administration. In someembodiments, the first level of fatty acid in the first sample isdetermined from the patient's medical history.

Another aspect of the present invention relates to a method of treatinga Her-2 related cancer comprising administering to a patient in needthereof an effective amount of a PARP inhibitor or metabolite thereof,wherein the PARP inhibitor or metabolite thereof inhibits a fatty acidsynthesis. Yet another aspect of the present invention relates to amethod of treating a Her-2 related cancer in a subject comprising: (i)determining a level of Her-2 expression in a sample from a subject, and(ii) administering an effective amount of a PARP inhibitor or metabolitethereof to the subject to inhibit fatty acid synthesis wherein theadministration is based on the determination of the level of Her-2expression, thereby treating the Her-2 related cancer in the subject.

In some embodiments of the aforementioned aspect of the presentinvention, the inhibition of the fatty acid synthesis comprisesinhibiting at least one enzyme of a glucose pathway or a fatty acidbiosynthetic pathway. In some embodiments, the inhibition of the fattyacid synthesis comprises inhibiting at least one enzyme of a fatty acidsynthase wherein the enzyme is selected from the group consisting ofacyl carrier protein, acetyl transferase, malonyl transferase,3-keto-acyl-ACP synthase, 3-ketoacyl-ACP reductase, 3-hydroxy-acyl-ACPdehydratase, and enoyl-ACP reductase. In some embodiments, the level ofHer-2 expression is up-regulated.

In some embodiments of the aforementioned aspect of the presentinvention, the PARP inhibitor or metabolite thereof is a compound offormula II, its pharmaceutically acceptable salts or prodrugs thereof:

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid.

In some preferred embodiments of the aforementioned aspect of thepresent invention, the PARP inhibitor is a compound of formula III, itspharmaceutically acceptable salts or prodrugs thereof:

In some embodiments of the aforementioned aspect of the presentinvention, the treatment is selected from the group consisting of oraladministration, transmucosal administration, buccal administration,nasal administration, inhalation, parental administration, intravenous,subcutaneous, intramuscular, sublingual, transdermal administration, andrectal administration. In some embodiments, the sample comprises acancer cell. In some embodiments, the method further comprisesadministering a Her-2 antibody. In some embodiments, the method furthercomprises surgery, radiation therapy, chemotherapy, gene therapy,immunotherapy, or a combination thereof.

Yet another aspect of the present invention relates to a method oftreating a metabolic disease, comprising administering to a patient inneed thereof an effective amount of a compound of formula II, itspharmaceutically acceptable salts or prodrugs thereof,

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid. In some preferred embodiments, thecompound of formula II, the pharmaceutically acceptable salts or theprodrugs thereof inhibits fatty acid synthesis, thereby treating themetabolic disease in the subject.

Yet another aspect of the present invention relates to a method oftreating a cancer in a subject comprising: (i) determining a level offatty acid in a sample from a subject, (ii) administering an effectiveamount of a compound of formula II, its pharmaceutically acceptablesalts or prodrugs thereof, to the subject wherein the administration isbased on the determination of the level of fatty acid, wherein thecompound of formula II, its pharmaceutically acceptable salts orprodrugs thereof comprises:

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid. In some preferred embodiments, thecompound of formula II, the pharmaceutically acceptable salts or theprodrugs thereof inhibit fatty acid synthesis, thereby treating thecancer in the subject.

In some embodiments of the aforementioned aspect of the presentinvention, the inhibition of the fatty acid synthesis comprisesinhibiting at least one enzyme of a glucose pathway or a fatty acidbiosynthetic pathway. In some embodiments, the inhibition of the fattyacid synthesis comprises inhibiting at least one enzyme of a fatty acidsynthase wherein the enzyme is selected from the group consisting ofacyl carrier protein, acetyl transferase, malonyl transferase,3-keto-acyl-ACP synthase, 3-ketoacyl-ACP reductase, 3-hydroxy-acyl-ACPdehydratase, and enoyl-ACP reductase. In some embodiments, the compoundof formula II, the pharmaceutically acceptable salts or the prodrugsthereof, comprises a compound of formula III, or a metabolite thereof:

In some embodiments of the aforementioned aspect of the presentinvention, the treatment is selected from the group consisting of oraladministration, transmucosal administration, buccal administration,nasal administration, inhalation, parental administration, intravenous,subcutaneous, intramuscular, sublingual, transdermal administration, andrectal administration. In some embodiments, the metabolic disease iscardiovascular, diabetes or obesity. In some embodiments, the cancer isfor example, Her-2 related cancer, colon adenocarcinoma, esophagusadenocarcinoma, liver hepatocellular carcinoma, squamous cell carcinoma,pancreas adenocarcinoma, islet cell tumor, rectum adenocarcinoma,gastrointestinal stromal tumor, stomach adenocarcinoma, adrenal corticalcarcinoma, follicular carcinoma, papillary, carcinoma, breast cancer,ductal carcinoma, lobular carcinoma, intraductal carcinoma, mucinouscarcinoma, phyllodes tumor, ovarian adenocarcinoma, endometriumadenocarcinoma, granulose cell tumor, mucinous cystadenocarcinoma,cervix adenocarcinoma, vulva squamous cell carcinoma, basal cellcarcinoma, prostate adenocarcinoma, giant cell tumor of bone, boneosteosarcoma, larynx carcinoma, lung adenocarcinoma, kidney carcinoma,urinary bladder carcinoma, Wilm's tumor, and lymphoma.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts metabolic flux of a glucose pathway.

FIG. 2 depicts pentose and futile cycles of a glucose pathway.

FIGS. 3A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit myristate synthesis in Hela cells.

FIGS. 4A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit palmitate synthesis in Hela cells.

FIGS. 5A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit stearate synthesis in Hela cells.

FIGS. 6A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit oleate synthesis in Hela cells.

FIGS. 7A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit C:22 fatty acid synthesis in Hela cells.

FIGS. 8A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit C:24 fatty acid synthesis in Hela cells.

FIGS. 9A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit arachidic acid synthesis in OVCAR-3 cells.

FIGS. 10A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit C:22 fatty acid synthesis in OVCAR-3 cells.

FIGS. 11A and B illustrate that 3 and 10 μM of 3-nitro-4-iodobenzamideinhibit C:24 fatty acid synthesis in OVCAR-3 cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term, “aryl” refers to optionally substituted mono- or bicyclicaromatic rings containing only carbon atoms. The term can also includephenyl group fused to a monocyclic cycloalkyl or monocycliccycloheteroalkyl group in which the point of attachment is on anaromatic portion. Examples of aryl groups include, e.g., phenyl,naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl,dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like.

The term, “heterocyclic” refers to an optionally substituted mono- orbicyclic aromatic ring containing at least one heteroatom (an atom otherthan carbon), such as N, O and S, with each ring containing about 5 toabout 6 atoms. Examples of heterocyclic groups include, e.g., pyrrolyl,isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl,thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl,triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl,benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.

The term “inhibit” or its grammatical equivalent, such as “inhibitory,”is not intended to require complete reduction in a biological activitybeing considered such as, synthesis of fatty acid. Such reduction ispreferably by at least about 50%, at least about 75%, at least about90%, and more preferably by at least about 95% of the activity of themolecule in the absence of the inhibitory effect, e.g., in the absenceof an inhibitor, such as PARP inhibitors disclosed in the invention.Most preferably, the term refers to an observable or measurablereduction in the synthesis of fatty acid. In treatment scenarios,preferably the inhibition is sufficient to produce a therapeutic and/orprophylactic benefit in the condition being treated.

The term “pharmaceutically acceptable salt” as used herein, means thosesalts which retain the biological effectiveness and properties of thecompounds of the present invention, and which are not biologically orotherwise undesirable.

The term “subject” or its grammatical equivalents as used herein refersto a warm-blooded animal such as a mammal who is healthy or is afflictedwith, or suspected to be afflicted with a disease. Preferably, “subject”refers to a human.

The term “treating” or its grammatical equivalents as used herein, meansachieving a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient can still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions can be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease can not have been made.

DEFINITIONS

“Nitrobenzamide precursor compound(s)” means a compound of the formula(Ia)

wherein R₁, R₂, R₃, R₄, and R₅ are, independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, (C₁-C₆)alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, and phenyl, wherein at leasttwo of the five R₁, R₂, R₃, R₄, and R₅ substituents are always hydrogen,at least one of the five substituents are always nitro, and at least onesubstituent positioned adjacent to a nitro is always iodo, andpharmaceutically acceptable salts, solvates, isomers, tautomers,metabolites, analogs, or prodrugs thereof. R₁, R₂, R₃, R₄, and R₅ canalso be a halide such as chloro, fluoro, or bromo. “Precursor compound”is a compound that undergoes one or more chemical or biochemicalprocesses (e.g., in a cell or in an organism) that result in ametabolite compound. The terms “precursor”, “precursor compound”,“benzamide precursor” or “nitrobenzamide precursor” are usedinterchangeably herein.

“Metabolite” means a compound produced through any in vitro or in vivometabolic process which results in a product that is different instructure than that of the starting compound. In other words, the term“metabolite” includes nitrobenzamide metabolite compounds. A metabolitecan include a varying number or types of substituents that are presentat any position relative to a precursor compound, such as the precursorcompound depicted in the formula (Ia). In addition, a metabolite canvary in the number of types of substituents that are present at anyposition relative to the compounds described herein. In addition, theterms “metabolite”, “metabolite compound”, “benzamide metabolitecompound” or “nitrobenzamide metabolite compound” are usedinterchangeably herein.

“Surgery” means any therapeutic or diagnostic procedure that involvesmethodical action of the hand or of the hand with an instrument, on thebody of a human or other mammal, to produce a curative, remedial, ordiagnostic effect.

“Radiation therapy” means exposing a patient to high-energy radiation,including without limitation x-rays, gamma rays, and neutrons. This typeof therapy includes without limitation external-beam therapy, internalradiation therapy, implant radiation, brachytherapy, systemic radiationtherapy, and radiotherapy.

“Chemotherapy” means the administration of one or more anti-cancer drugsand/or other agents to a cancer patient by various methods, includingintravenous, oral, intramuscular, intraperitoneal, intravesical,subcutaneous, transdermal, buccal, or inhalation or in the form of asuppository. Chemotherapy may be given prior to surgery to shrink alarge tumor prior to a surgical procedure to remove it, after surgery orradiation therapy to prevent the growth of any remaining cancer cells inthe body.

The terms “effective amount” or “pharmaceutically effective amount”refer to a non-toxic but sufficient amount of the agent to provide thedesired biological, therapeutic, and/or prophylactic result. That resultcan be reduction and/or alleviation of the signs, symptoms, or causes ofa disease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of anitrobenzamide metabolite compound as disclosed herein per se or acomposition comprising the nitrobenzamide metabolite compound hereinrequired to result in a clinically significant decrease in a disease. Anappropriate effective amount in any individual case may be determined byone of ordinary skill in the art using routine experimentation.

The term “PARP inhibitor or metabolite thereof” includes compounds thatare themselves PARP inhibitors as well as the active metabolites ofthose compounds. In some embodiments, said metabolites of PARPinhibitors are themselves PARP inhibitors, whether isolated or not. Isome embodiments, said metabolites of PARP inhibitors are isolated andpurified to a purity of at least 50%, at least 55%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.9% or purer, before beingcombined with one or more pharmaceutically acceptable ingredients tomake a pharmaceutically acceptable dosage form as described in moredetail herein. Metabolites of PARP inhibitors are metabolized forms ofPARP inhibitor “precursor compounds” or “precursors,” which aredescribed in more detail herein.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

The term “treating” and its grammatical equivalents as used hereininclude achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. For example, in a cancer patient,therapeutic benefit includes eradication or amelioration of theunderlying cancer. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the patient, notwithstanding the fact that the patient maystill be afflicted with the underlying disorder. For prophylacticbenefit, a method of the invention may be performed on, or a compositionof the invention administered to a patient at risk of developing cancer,or to a patient reporting one or more of the physiological symptoms ofsuch conditions, even though a diagnosis of the condition may not havebeen made.

As used herein “BA” means 4-iodo-3-nitrobenzamide; “BNO” means4-iodo-3-nitrosobenzamide; “BNHOH” means 4-iodo-3-hydroxyaminobenzamide.

Nitrobenzamide Metabolite Compounds

PARP inhibitors (Precursor compounds) useful in the present inventionare of Formula (Ia)

wherein R₁, R₂, R₃, R₄, and R₅ are, independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, (C₁-C₆)alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, and phenyl, wherein at leasttwo of the five R₁, R₂, R₃, R₄, and R₅ substituents are always hydrogen,at least one of the five substituents are always nitro, and at least onesubstituent positioned adjacent to a nitro is always iodo, andpharmaceutically acceptable salts, solvates, isomers, tautomers,metabolites, analogs, or pro-drugs thereof. R₁, R₂, R₃, R₄, and R₅ canalso be a halide such as chloro, fluoro, or bromo substituents.

A preferred (PARP inhibitor) precursor compound of formula Ia is:

Metabolites of PARP inhibitors useful in the present invention are ofthe Formula (IIa):

wherein either: (1) at least one of R₁, R₂, R₃, R₄, and R₅ substituentis always a sulfur-containing substituent, and the remainingsubstituents R₁, R₂, R₃, R₄, and R₅ are independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, bromo,fluoro, chloro, (C₁-C₆) alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, andphenyl, wherein at least two of the five R₁, R₂, R₃, R₄, and R₅substituents are always hydrogen; or (2) at least one of R₁, R₂, R₃, R₄,and R₅ substituents is not a sulfur-containing substituent and at leastone of the five substituents R₁, R₂, R₃, R₄, and R₅ is always iodo, andwherein said iodo is always adjacent to a R₁, R₂, R₃, R₄, or R₅ groupthat is either a nitro, a nitroso, a hydroxyamino, hydroxy or an aminogroup; and pharmaceutically acceptable salts, solvates, isomers,tautomers, metabolites, analogs, or pro-drugs thereof. In someembodiments, the compounds of (2) are such that the iodo group is alwaysadjacent a R₁, R₂, R₃, R₄ or R₅ group that is a nitroso, hydroxyamino,hydroxy or amino group. In some embodiments, the compounds of (2) aresuch that the iodo the iodo group is always adjacent a R₁, R₂, R₃, R₄ orR₅ group that is a nitroso, hydroxyamino, or amino group. In someembodiments, the sulfur-containing substituent is —SR₆, wherein R₆ isselected from the group consisting of hydrogen, C₁-C₆ acyl, optionallysubstituted (C₁-C₆) alkyl, optionally substituted (C₃-C₇) cycloalkyl,optionally substituted (C₃-C₇) heterocyclic and optionally substitutedaryl.

The following compositions are some preferred PARP inhibitor metabolitecompounds embraced by Formula IIa, each represented by a chemicalformula:

While not being limited to any one particular mechanism, the followingprovides an example for MS292 metabolism via a nitroreductase orglutathione conjugation mechanism:

Nitroreductase Mechanism

BA glutathione conjugation and metabolism:

The present invention provides for the use of the aforesaidnitrobenzamide metabolite compounds for the treatment of other breastcancers including a ductal carcinoma in a mammary gland, other forms ofleukemia including acute pro-myelocytic leukemia in peripheral blood,ovarian cancer, lung cancer, bladder cancer, prostate cancer, pancreaticcancer, and cervical cancer, as well as other cancer types describedherein.

It has been reported that nitrobenzamide metabolite compounds haveselective cytotoxicity upon malignant cancer cells but not uponnon-malignant cancer cells. See Rice et at., Proc. Natl. Acad. Sci. USA89:7703-7707 (1992). In one embodiment, the nitrobenzamide metabolitecompounds utilized in the methods of the present invention may exhibitmore selective toxicity towards tumor cells than non-tumor cells.

One aspect of the present invention provides methods of treating fattyacid synthesis related diseases by administering an effective amount ofa poly(ADP)ribose polymerase enzyme (PARP) inhibitor wherein the PARPinhibitor inhibits fatty acid synthesis. Fatty acid synthesis relatediseases include, but are not limited to cancer, diabetes, obesity,Niemann-Pick disease, Gaucher's disease, metachromatic leukodystrophy,Fabry's disease, hyaline membrane disease, Tay-Sachs disease, Sandhoffdiseases, Krabbe's disease, fucosidosis, sulfatide lipodosis, andFarber's lipogranulomatosis. The fatty acid synthesis related diseasesincludes diseases with abnormality in fatty acid metabolism.

Fatty acid synthesis is also related to inflammation (predominantlythose of the joints, skin and eyes), reproductive function (includingthe induction of labor), inhibiting gastric acid secretion, regulatingblood pressure through vasodilation or constriction, and inhibiting oractivating platelet aggregation and thrombosis. In some preferredembodiments, the fatty acid synthesis related diseases include obesity,diabetes, or cardiovascular disease.

Another aspect of the present invention relates to methods for treatmentof cancer in a subject by administering an effective amount of a PARPinhibitor or metabolite thereof wherein the PARP inhibitor or metabolitethereof inhibits fatty acid synthesis. The methods particularly relateto treating a cancer in a subject by identifying a level of a fatty acidin a sample of a subject, and administering an effective amount of aPARP inhibitor or metabolite thereof to inhibit fatty acid synthesiswhere the administration is based on the level of fatty acid, therebytreating the cancer in the subject.

The test sample can be used directly as obtained from the source orfollowing a pretreatment to modify the character of the sample. Thesample can be derived from any biological source, such as tissues orextracts, including cells, and physiological fluids, such as, forexample, whole blood, plasma, serum, saliva, ocular lens fluid,cerebrospinal fluid, sweat, urine, milk, ascots fluid, synovial fluid,peritoneal fluid and the like. The sample is obtained from animals orhumans, preferably from humans. The sample can be treated prior to use,such as preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treating a sample can involve filtration, distillation,extraction, concentration, inactivation of interfering components, theaddition of reagents, and the like.

Typically, in normal humans, the fatty acid biosynthetic pathway isdown-regulated and most of the fatty acid in the body comes from dietaryfat. However, in humans suffering from cancer, fatty acid biosyntheticpathway is up-regulated resulting in elevated levels of fatty acid inthe tumor tissues. The up-regulated fatty acid synthesis in variouscancers suggests that fatty acid synthesis provides an advantage fortumor growth. The inhibition of fatty acid synthesis can result indepletion of the membrane lipids, which can cause cell death (Khaddar etal. Proc. Natl. Acad. Sic. 1994, vol. 91, p. 6379-6383). The presentinvention provides treatment of cancer with an effective amount of aPARP inhibitor or metabolite thereof wherein the PARP inhibitor ormetabolite thereof inhibits the fatty acid synthesis. Various cancersinclude but are not limited to, bladder, breast, colon, rectum,prostate, ovary, salivary gland, skin adnexae, bile duct, endocervix,ectocervix, vagina, esophagus, nasopharynx, oropharynx, or those of germcell origin, carcinomas or adenocarcinomas of the stomach, endometrium,kidney, liver and lung, melanoma and mesothelioma.

In some embodiments of the present invention, the method includestreatment of Her-2 related cancer by administering an effective amountof a PARP inhibitor or metabolite thereof wherein the PARP inhibitor ormetabolite thereof inhibits a fatty acid synthesis. Not intending tolimit the scope of the present invention, the fatty acid synthase(FAS)-catalyzed de novo fatty acid biosynthesis may contribute to thecancer phenotype by virtue of its ability to specifically regulate theexpression and activity of Her-2/neu (erbB-2) oncogene (Menendez J A etal. Drug News Perspect. 2005, 18(6), 375-85; Menendez J A et al. J. CellBiochem. 2005, 94(5), 857-63). The inhibition of the fatty acidsynthesis by the PARP inhibitor or metabolite thereof can result intreatment of Her-2 related cancer.

In some embodiments of the present invention, the method of treatment ofHer-2 related cancer includes determining a level of Her-2 expression ina sample from a subject and administering an effective amount of a PARPinhibitor or metabolite thereof to the subject to inhibit fatty acidsynthesis wherein the administration is based on the determination ofthe level of Her-2 expression in the sample from the subject. In somepreferred embodiments, the sample comprises a cancer cell. If the levelof Her-2 expression in the sample is higher then the patient is treatedwith an effective amount of the PARP inhibitor or metabolite thereof.Without limiting the scope of the present invention the PARP inhibitoror metabolite thereof inhibits fatty acid synthesis and thereby treats aHer-2 related cancer. In some embodiments, the PARP inhibitor ormetabolite thereof inhibits fatty acid synthesis by inhibiting one ormore enzymes of a glucose pathway or a fatty acid biosynthetic pathway.In some embodiments, the PARP inhibitor or metabolite thereof isadministered in combination with a Her-2 antibody. An example of a Her-2antibody is herceptin.

In some preferred embodiments, the methods include treatment of obesityby administering an effective amount of a PARP inhibitor or metabolitethereof wherein the PARP inhibitor or metabolite thereof inhibits afatty acid synthesis. Obesity is a major health problem that is becomingmore common among adults and increasing rapidly in children andadolescents. Obesity has been linked to a broad range of physical,emotional and socioeconomic problems. The method of the presentinvention for reducing obesity, are applicable to humans and otheranimals including vertebrates, especially mammals. Animals includepoultry, swine, cattle, sheep, and other animals where reduction in fataccumulation without reduction in muscle mass can be desirable forveterinary health or economic reasons. The PARP inhibitors can beadministered in accordance with the methods of the present invention todogs, cats, horses and other animals for veterinary health reasons.

In some preferred embodiments of the present invention the methodsinclude treatment of cardiovascular disease by administering aneffective amount of a PARP inhibitor or metabolite thereof wherein thePARP inhibitor or metabolite thereof inhibits a fatty acid synthesis. Insome people excess body fat causes an increased risk for vasculardisease, including heart disease and stroke. Increased fat stored inintra-abdominal deposits can be associated with, and can cause, anincrease in risk factors for atherosclerotic cardiovascular disease(ASCVD). The risk factors for ASCVD include: hypertension, elevatedlevels of cholesterol, particularly LDL-cholesterol, in the blood,diabetes and hyperglycemia. People with hypertension are likely todevelop impaired glucose tolerance, a type of pre-diabetes.

In some preferred embodiments, the methods include treatment of diabetesby administering an effective amount of a PARP inhibitor or metabolitethereof wherein the PARP inhibitor or metabolite thereof inhibits afatty acid synthesis. Patients suffering from diabetes can have any ofseveral lipid abnormalities. Common lipid profiles in the patient withdiabetes include, elevated levels of triglycerides, low-densitylipoproteins (LDL), and very low-density lipoproteins (VLDL), along withlower than normal levels of high-density lipoprotein (HDL). The combinedeffect of these factors results in promotion of atherosclerosis andthrombosis. Other diseases include, but are not limited to,hyperinsulinemia, insulin resistance, myocardial infarction, fattyliver, polycystic ovarian syndrome, hemochromatosis, non-alcoholicsteatohepatitis, diabetic kidney disease, etc.

While it is preferred that the level of the fatty acid in a subject isdetermined prior to the treatment with PARP inhibitors or metabolitesthereof, the skilled clinician will recognize that such determination isnot always necessary. The reduction of the tumor burden after thetreatment of a cancer patient with a PARP inhibitor or metabolitethereof would demonstrate the presence of elevated levels of fatty acidin the tumor before the treatment. Where a cancer patient can besuccessfully treated by the method of this invention, independentdetermination of a level of fatty acid in a subject can be unnecessary.Such empirical treatment of cancer of the type usually found to expresselevated levels of fatty acid is also within the scope of the presentinvention.

Another aspect of the present invention relates to a method ofmonitoring a therapeutic effectiveness of a PARP inhibitor or metabolitethereof in a treatment of a disease by administering an effective amountof a PARP inhibitor or metabolite thereof to a patient to inhibit fattyacid synthesis; comparing a first and a second level of fatty acid in afirst and second sample from the patient wherein the first level and thefirst sample are obtained prior to administration of the PARP inhibitoror metabolite thereof and the second level and the second sample areobtained after administration of the PARP inhibitor or metabolitethereof; and determining a therapeutic effectiveness of the PARPinhibitor or metabolite thereof in a treatment of a disease in thepatient based on the comparison.

The first level of the fatty acid from the first sample can bedetermined just before the administration of the PARP inhibitor ormetabolite thereof to the patient or can be obtained day/s before,week's before, month/s before, or year/s before the administration ofthe PARP inhibitor or metabolite thereof to the patient. The first levelfrom first sample can be obtained from a medical history of the patient.The second level of the fatty acid from the second sample can be foundto be lower than the first level of the fatty acid from the first samplethereby indicating the inhibition of the fatty acid synthesis in thepatient by the PARP inhibitor or metabolite thereof and hence indicatingthe therapeutic effectiveness of the PARP inhibitor or metabolitethereof. The second level of the fatty acid from the second sample canbe found to be higher than the first level of the fatty acid from thefirst sample and may thereby indicate the less therapeutic effectivenessof the PARP inhibitor or metabolite thereof. Such monitoring of thetherapeutic effectiveness of the PARP inhibitor or metabolite thereofcan be useful in adjusting the dosage and personalizing the dosingregimen for the patient. Such monitoring can also be used in clinicaltrials.

The inhibition of fatty acid synthesis in the methods of the presentinvention can involve inhibition of one or more enzymes of a glucosepathway or a fatty acid biosynthetic pathway. Without limiting the scopeof the present invention, various steps and the enzymes involved in theglucose pathway and the fatty acid biosynthetic pathway are disclosedherein. Any one or more of the steps and/or the enzymes can be inhibitedby the PARP inhibitors or metabolites thereof of the present invention.

Glucose Pathway

Oxidation of glucose is known as glycolysis where glucose is oxidized toeither lactate or pyruvate. Under aerobic conditions, the product inmost tissues is pyruvate and the pathway is known as aerobic glycolysis.When oxygen is depleted, as for instance during prolonged vigorousexercise, the dominant glycolytic product in many tissues is lactate andthe process is known as anaerobic glycolysis. The pathway of glycolysiscan be seen as consisting of 2 separate phases. In the first phase, 2equivalents of ATP are used to convert glucose to fructose1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded topyruvate, with the production of 4 equivalents of ATP and 2 equivalentsof NADH (see FIGS. 1 and 2).

The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate(G6P) is the first reaction of glycolysis, and is catalyzed bytissue-specific isoenzymes known as hexokinases. Four mammalian isozymesof hexokinase are known (types I-IV), with the type IV isozyme oftenreferred to as glucokinase. Glucokinase is the form of the enzyme foundin hepatocytes. The regulation of hexokinase and glucokinase activitiesis also different. Hexokinases I, II, and III are allostericallyinhibited by product (G6P) accumulation, whereas glucokinases are not.The latter further insures liver accumulation of glucose stores duringtimes of glucose excess, while favoring peripheral glucose utilizationwhen glucose is required to supply energy to peripheral tissues.

The second reaction of glycolysis is an isomerization, in which G6P isconverted to fructose 6-phosphate (F6P). The enzyme catalyzing thisreaction is phosphohexose isomerase (also known as phosphoglucoseisomerase). The next reaction of glycolysis involves the utilization ofa second ATP to convert F6P to fructose 1,6-bisphosphate (F1,6BP). Thisreaction is catalyzed by 6-phosphofructo-1-kinase, better known asphosphofructokinase-1 or PFK-1. Fructose units readily flow in thereverse (gluconeogenic) direction because of the ubiquitous presence ofthe hydrolytic enzyme, fructose-1,6-bisphosphatase (F-1,6-BPase). Thepresence of these two enzymes in the same cell compartment provides anexample of a metabolic futile cycle, which if unregulated would rapidlydeplete cell energy stores. However, the activity of these two enzymesis so highly regulated that PFK-1 is considered to be the rate-limitingenzyme of glycolysis and F-1,6-BPase is considered to be therate-limiting enzyme in gluconeogenesis.

Aldolase catalyses the hydrolysis of F1,6BP into two 3-carbon products:dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P).The aldolase reaction proceeds readily in the reverse direction, beingutilized for both glycolysis and gluconeogenesis. The two products ofthe aldolase reaction equilibrate readily in a reaction catalyzed bytriose phosphate isomerase. Succeeding reactions of glycolysis utilizeG3P as a substrate; thus, the aldolase reaction is pulled in theglycolytic direction by mass action principals.

The second phase of glucose catabolism features the energy-yieldingglycolytic reactions that produce ATP and NADH. In the first of thesereactions, glyceraldehyde-3-P dehydrogenase (G3PDH) catalyzes theNAD⁺-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) andNADH. The G3PDH reaction is reversible, and the same enzyme catalyzesthe reverse reaction during gluconeogenesis.

The high-energy phosphate of 1,3-BPG is used to form ATP and3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase.Associated with the phosphoglycerate kinase pathway is an importantreaction of erythrocytes, the formation of 2,3-bisphosphoglycerate,2,3BPG by the enzyme bisphosphoglycerate mutase. 2,3BPG is an importantregulator of hemoglobin's affinity for oxygen. The2,3-bisphosphoglycerate phosphatase degrades 2,3BPG to3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3BPGshunt thus operates with the expenditure of 1 equivalent of ATP pertriose passed through the shunt.

The remaining reactions of glycolysis are aimed at converting the 3PG to2PG by phosphoglycerate mutase and the 2PG conversion tophosphoenoylpyruvate (PEP) is catalyzed by enolase. The final reactionof aerobic glycolysis is catalyzed by the highly regulated enzymepyruvate kinase (PK). In this exergonic reaction, the high-energyphosphate of PEP is conserved as ATP. The loss of phosphate by PEP leadsto the production of pyruvate in an unstable enol form, whichspontaneously tautomerizes to the more stable, keto form of pyruvate.Under anaerobic conditions and in erythrocytes under aerobic conditions,pyruvate is converted to lactate by the enzyme lactate dehydrogenase(LDH), and the lactate is transported out of the cell into thecirculation.

Under aerobic conditions, pyruvate in most cells is further metabolizedvia the TCA cycle. Pyruvate is preferentially oxidized to CO₂ and H₂O inthe TCA cycle. When transported into the mitochondrion, pyruvateencounters two principal metabolizing enzymes: pyruvate carboxylase (agluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzymeof the PDH complex. When the energy charge is low, CoA is not acylated,pyruvate carboxylase is inactive, and pyruvate is preferentiallymetabolized via the PDH complex and the enzymes of the TCA cycle to CO₂and H₂O. The PDH complex is comprised of multiple copies of 3 separateenzymes: pyruvate dehydrogenase (20-30 copies), dihydrolipoyltransacetylase (60 copies) and dihydrolipoyl dehydrogenase (6 copies).The complex also requires 5 different coenzymes: CoA, NAD⁺, FAD⁺, lipoicacid and thiamine pyrophosphate (TPP). The net result of the reactionsof the PDH complex are:

Pyruvate+CoA+NAD⁺→CO₂+acetyl-CoA+NADH+H⁺

The reactions of the PDH complex serve to interconnect the metabolicpathways of glycolysis, gluconeogenesis and fatty acid synthesis to theTCA cycle.

Fatty Acid Biosynthetic Pathway

Fatty acids are synthesized by fatty acid synthase (FAS) using thesubstrates acetyl CoA, malonyl CoA and NADPH. Thus, the fatty acidsynthesis pathway is usually considered to involve four enzymes, FAS andthe three enzymes which produce its substrates: acetyl CoA carboxylase(ACC), malic enzyme and citrate lyase. Other enzymes which can feedsubstrates into the pathway, such as the enzymes which produce NADPH viathe hexose monophosphate shunt, can also affect the rate of fatty acidsynthesis, and thus in cells that depend on endogenously synthesizedfatty acid Inhibition of the expression or the activity of any of theseenzymes can affect growth of cancer cells that are dependent onendogenously synthesized fatty acid.

The synthesis of malonyl-CoA is the first step of fatty acid synthesisand the enzyme that catalyzes this reaction, acetyl-CoA carboxylase(ACC), is the site of regulation of fatty acid synthesis. Like otherenzymes that transfer CO₂ to substrates, ACC requires a biotinco-factor. The synthesis of fatty acids from acetyl-CoA and malonyl-CoAis carried out by fatty acid synthase, FAS. The active enzyme is a dimerof identical subunits.

All of the reactions of fatty acid synthesis are carried out by themultiple enzymatic activities of FAS. Like fat oxidation, fat synthesisinvolves 4 enzymatic activities. These are β-keto-ACP synthase,β-keto-ACP reductase, 3-OH acyl-ACP dehydratase and enoyl-CoA reductase.The two reduction reactions require NADPH oxidation to NADP⁺. Theprimary fatty acid synthesized by FAS is palmitate.

Palmitate is a 16:0 fatty acid, i.e. 16 carbons and no sites ofunsaturation. Palmitate is then released from the enzyme and can thenundergo separate elongation and/or unsaturation to yield other fattyacid molecules. Elongation and unsaturation of fatty acids occurs inboth the mitochondria and endoplasmic reticulum (microsomal membranes).The predominant site of these processes is in the ER membranes.Elongation involves condensation of acyl-CoA groups with malonyl-CoA.The resultant product is two carbons longer (CO₂ is released frommalonyl-CoA as in the FAS reaction) which undergoes reduction,dehydration and reduction yielding a saturated fatty acid. The reductionreactions of elongation require NADPH as co-factor just as for thesimilar reactions catalyzed by FAS. Mitochondrial elongation involvesacetyl-CoA units and is a reversal of oxidation except that the finalreduction utilizes NADPH instead of FADH₂ as co-factor.

Desaturation occurs in the ER membranes as well and in mammalian cellsand involves 4 broad specificity fatty acyl-CoA desaturases (non-hemeiron containing enzymes). These enzymes introduce unsaturation at C₄,C₅, C₆ or C₉. The electrons transferred from the oxidized fatty acidsduring desaturation are transferred from the desaturases to cytochromeb5 and then NADH-cytochrome b5 reductase. Some of the saturated fattyacids include, but are not limited to, lauric acid (C:12), myristic acid(C:14), palmitic acid (C:16), stearic acid (C:18), oleic acid (C:18-1),arachidic acid (C:20), C:22 and C:24. Some of the unsaturated acidsinclude, but are not limited to, docosahexaenoic acid, eicosapentaenoicacid, arachidonic acid, oleic acid, and erucic acid.

Analysis Techniques

The determination of the level of fatty acid in a sample of a subject orthe determination of the inhibition of fatty acid synthesis aftertreatment with PARP inhibitor or metabolite thereof can involve variousdetection techniques known in the art including but not limited to,enzyme assays, mass spectrometry such as gas chromatography/massspectrometry (GC/MS), mass selective detector analysis (MSD), chemicalionization and selected monitoring (SIM), mass isotopomer distributionanalysis (MIDA), high performance liquid chromatography (HPLC) ornuclear magnetic resonance (NMR).

The determination of inhibition of fatty acid synthesis after treatmentof a subject with an effective amount of a PARP inhibitor or metabolitethereof can involve analyzing various metabolites or the molecularfluxes of the glucose or the fatty acid biosynthetic pathway in a samplefrom the subject. The determination can involve one or more comparisonswith reference samples. The reference samples are typically obtainedfrom the same subject or from a different subject who is either notaffected with the disease (such as, normal subject) or is a patient. Thereference sample could be obtained from one subject, multiple subjectsor could be synthetically generated. The identification can also involvethe comparison of the identification data with the databases. Oneembodiment of the invention relates to identifying the level of fattyacid in a subject afflicted with a disease, such as cancer andcorrelating it with the fatty acid level of the normal subjects. If thelevel of the fatty acid is up-regulated in the subject afflicted withthe disease then the subject is treated with an effective amount of PARPinhibitor or metabolite thereof that inhibits the synthesis of the fattyacid.

In some embodiments, the step of comparison the level of fatty acid isperformed by a software algorithm. Preferably, the data generated istransformed into computer readable form; and an algorithm is executedthat classifies the data according to user input parameters, fordetecting signals that represent level of fatty acid in diseasedpatients and level of fatty acid in normal subjects.

The metabolites of the glucose or the fatty acid biosynthetic pathwaythat can be analyzed in the methods of the present invention include butare not limited to, fatty acid or its metabolites, enzymes, glucose,glutamate, glycogen, lactate, CO₂, acetyl Co-A, RNA ribose and DNAdeoxyribose. The molecular fluxes of the glucose or fatty acidbiosynthetic pathway include, but are not limited to, glucose uptakefrom culture media; lactate production from glucose (anaerobicglycolysis); ¹³CO₂ release from glucose via TCA cycle; TCA cycleanaplerotic flux; glycogen synthesis; de novo fatty acid synthesis,elongation, desaturation and acetyl-CoA synthesis; and pentose cycle-RNAand DNA ribose synthesis via oxidative and non-oxidative reactions. Themetabolites can be chemically derivatized after extraction and beforeanalysis. The analyzing techniques are well known in the art and arewithin the scope of the present invention.

The assay techniques include activity assays or stains, immunoassaysusing antibodies, assays measuring enzyme mRNA such as FAS mRNA, and thelike. Expression of the enzymes involved in the glucose pathway or thefatty acid biosynthetic pathway can be determined directly in tumortissue samples obtained through procedures such as biopsies, resectionsor needle aspirates, using assays such as immunohistochemistry, cytosolenzyme immunoassay or radioimmunoassay, in situ hybridization of nucleicacid probes with mRNA targets, or direct measurement of enzyme activity.Expression of the enzymes can be indirectly measured in biological fluidsamples obtained from subjects, such as blood, urine, serum, lymph,saliva, semen, ascites, or especially plasma, using any suitable assays.Preferred assays include enzyme immunoassay or radioimmunoassay.

In some embodiments, the therapeutic effect of a PARP inhibitor ormetabolite thereof on a cell, tissue, or organism can be determined byanalyzing the rates of synthesis or removal of a plurality ofmetabolites such as fatty acids in the cell, tissue, or organism afterthe administration of an effective amount of the PARP inhibitor ormetabolite thereof. By this method, the inhibition of the fatty acidsynthesis by the PARP inhibitor or metabolite thereof can be monitored.

In some preferred embodiments of the present invention, the metabolitesor the molecular fluxes of the glucose or the fatty acid biosyntheticpathway are analyzed by mass isotopomer distribution analysis (MIDA). Insome embodiments, a molecular flux rate of a plurality of metabolites inall or a portion of a cell, tissue or organism is analyzed. One or moreisotope-labeled metabolites or metabolite precursors are administered tothe cell, tissue or organism for a period of time sufficient for one ormore isotope labels to be incorporated into a plurality of metabolitesin the cell, tissue or organism. A media or a cell pellet is collectedfrom the cell, tissue, or organism. A plurality of mass isotopomericenvelopes of ions representing individual metabolites in the cell pelletor the media are identified by mass spectrometry. In addition, therelative and absolute mass isotopomer abundances of the ions within theisotopic envelopes corresponding to each identified metabolite arequantified by mass spectrometry. These relative and absolute massisotopomer abundances allow the rates of synthesis or removal of eachidentified metabolite to be calculated, and the molecular flux rates ofthe plurality of metabolites thereby to be determined. In someembodiments, the metabolites can be derivatized prior to introductioninto the mass spectrometer. The derivatization can be any method knownin the art, such as biochemically degrading the metabolites orchemically altering the metabolites.

Isotopic labels include specific heavy isotopes of elements, present inbiomolecules, such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³⁵S, ³⁴S, or can contain otherisotopes of elements present in biomolecules, such as ³H, ¹⁴C, ³⁵S,¹²⁵I, ¹³¹I. Isotope labeled metabolites include but are not limited to,²H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeled amino acids, ¹³C-labeled aminoacids, ¹⁵N-labeled amino adds, ¹⁸O-labeled amino acids, ³³S or³⁴S-labeled amino acids, ³H₂O, ³H-labeled amino adds, ¹⁴C-labeled aminoacids, ¹⁴CO₂, and H¹⁴CO₂ etc.

Metabolic flux estimation deals with uncovering the steady-statevelocities of biochemical reactions in vivo. The knowledge of the fluxeshelps in the optimization of the metabolic pathways towards high yieldof the product metabolite of interest. The use of ¹³C-labeled substrateis a method to quantify intracellular fluxes of the cell when there ismore than one alternative pathway between two metabolites. In thepresent invention, [1, 2-¹³C₂]-D-glucose is used in the media as theonly source of glucose. The tracing methods are based on measuring theisotopomer distributions, that is, the different ¹³C-isotopic versionsof the metabolite with their relative abundances. By comparing themeasured isotopomer distribution to the distributions expected whenusing each alternative pathway, the information about the distributionof the fluxes among the pathways can be deduced.

There are two methods to determine the positional isotopomerdistribution of the atoms, nuclear magnetic resonance (NMR) and massspectrometry (MS). MS can be coupled with liquid (LC-MS) or gas (GC-MS)chromatographic separation. The methods also can be combined to gainmore information about the positional isotopomer distribution and thusabout the fluxes as well. In some preferred embodiments of the presentinvention, the isotopomer distribution of the atoms and the metabolicfluxes are determined by mass spectrometry. The mass spectrometricmethod that can be used is a GC-MS with electron impact (EI) ionizationand a full scan mode. The mass isotopomers of the metabolite moleculefragment simultaneously in the ionization chamber because of highenergy, and a set of fragment ions are observed in the spectrum. WhenGC-tandem mass spectrometry method (GC-MS/MS) is used, the isotopomerion of interest can be chosen and fragmented (daughter ion scanning) toget an additional information. Many of the metabolites analyzed arepolar, so derivatization may be needed to convert the metabolites to bevolatile enough for GC-MS analysis. In isotopomer distributioncalculations, the atoms from dramatizing reagent are also taken intoaccount. With an LC-MS method, when an electrospray ionization (ESI) isused for analyzing polar metabolites, usually no derivatization may beneeded.

The extent to which an isotopomer distribution is identifiable fromtandem mass spectrometric data can depend on two aspects: first, thefrequencies of mass isotopomers may need to be sufficient for thecorresponding peaks to be reliably detected. Second, the fragmentationof the molecule may need to be sufficient. In order to completelypinpoint the abundance of each isotopomer, for every pair of carbonsthere may need to be a fragment where exactly one of the carbonsappears.

Treatment with PARP Inhibitors or Metabolites Thereof.

The compounds suitable for use in the present invention are compoundsthat inhibit fatty acid synthesis. Preferably the inhibitors (andmetabolites thereof) are PARP inhibitors. The inhibition of fatty acidsynthesis by the compounds as provided herein, can involve inhibition ofone or more enzymes of a glucose pathway or a fatty acid biosyntheticpathway. A preferred PARP inhibitor of the present invention is acompound of formula I, its pharmaceutically acceptable salts or prodrugsthereof:

wherein, R₁, R₂, R₃, R₄, and R₅ are independently selected from a groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and a sulfur-containing moiety. In some embodiments,the sulfur containing moiety is —SR₆, wherein R₆ is selected from thegroup consisting of hydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆)alkyl, optionally substituted (C₃-C₇) cycloalkyl, optionally substituted(C₃-C₇) heterocyclic and optionally substituted aryl.

Another preferred PARP inhibitor is compound of formula II, itspharmaceutically acceptable salts or prodrugs thereof:

wherein, R₁, R₃, R₄, and R₅ are independently selected from a groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents. In some embodiments R₆ is an optionally substituted(C₁-C₆) alkyl, which is a residue of an S-linked cysteine moiety, whichmay be a single cysteine amino acid or may form part of a dipeptide,tripeptide, tetrapeptide, pentapeptide or higher-order peptidecontaining cysteine as an amino acid.

A preferred PARP inhibitor is 3-nitro-4-iodobenzamide of formula III,its pharmaceutically acceptable salts or prodrugs thereof:

Some embodiments of the present invention relate to a method of treatinga metabolic disease by administering to a patient in need thereof aneffective amount of a compound of formula II, its pharmaceuticallyacceptable salts or prodrugs thereof,

wherein, R₁, R₃, R₄, and R₅ are independently selected from the groupconsisting of hydrogen, hydroxy, optionally substituted amine, carboxyl,ester, nitroso, nitro, halogen, optionally substituted (C₁-C₆) alkyl,optionally substituted (C₁-C₆) alkoxy, optionally substituted (C₃-C₇)cycloalkyl, optionally substituted (C₃-C₇) heterocyclic, optionallysubstituted aryl and —SR₆; R₂ is either nitro or nitroso; and wherein atleast two of the R₁, R₃, R₄, and R₅ substituents are always hydrogen;wherein R₆ is —SR₆, wherein R₆ is selected from the group consisting ofhydrogen, C₁-C₆ acyl, optionally substituted (C₁-C₆) alkyl, optionallysubstituted (C₃-C₇) cycloalkyl, optionally substituted (C₃-C₇)heterocyclic and optionally substituted aryl and the optionalsubstituents; and wherein the compound of formula II, itspharmaceutically acceptable salts or prodrugs thereof inhibits fattyacid synthesis, thereby treating the metabolic disease in the subject.In some embodiments R₆ is an optionally substituted (C₁-C₆) alkyl, whichis a residue of an S-linked cysteine moiety, which may be a singlecysteine amino acid or may form part of a dipeptide, tripeptide,tetrapeptide, pentapeptide or higher-order peptide containing cysteineas an amino acid.

The present invention further contemplates the use of metabolites of thecompounds of formula I. Some metabolites useful in the present inventionare of the Formula (IIa):

wherein either: (1) at least one of R₁, R₂, R₃, R₄, and R₅ substituentis always a sulfur-containing substituent, and the remainingsubstituents R₁, R₂, R₃, R₄, and R₅ are independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, bromo,fluoro, chloro, (C₁-C₆) alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, andphenyl, wherein at least two of the five R₁, R₂, R₃, R₄, and R₅substituents are always hydrogen; or (2) at least one of R₁, R₂, R₃, R₄,and R₅ substituents is not a sulfur-containing substituent and at leastone of the five substituents R₁, R₂, R₃, R₄, and R₅ is always iodo, andwherein said iodo is always adjacent to a R₁, R₂, R₃, R₄, or R₅ groupthat is either a nitro, a nitroso, a hydroxyamino, hydroxy or an aminogroup; and pharmaceutically acceptable salts, solvates, isomers,tautomers, metabolites, analogs, or pro-drugs thereof. In someembodiments, the compounds of (2) are such that the iodo group is alwaysadjacent a R₁, R₂, R₃, R₄ or R₅ group that is a nitroso, hydroxyamino,hydroxy or amino group. In some embodiments, the compounds of (2) aresuch that the iodo the iodo group is always adjacent a R₁, R₂, R₃, R₄ orR₅ group that is a nitroso, hydroxyamino, or amino group.

The following compositions are preferred metabolite compounds, eachrepresented by a chemical formula:

In some embodiments, the metabolites of the compounds as provided hereinmay be used in the methods of the present invention. For example, themetabolites include as described in U.S. application entitled,“Treatment of Cancer”; inventors Ernest Kun, Jerome Mendeleyev, CarolBasbaum, Hassan Lemjabbar-Alaoui, and Valeria Ossovskaya; filed on Sep.5, 2006; Attorney docket number 28825-729.101, incorporated herein byreference in its entirety.

Some embodiments of the present invention relate to a method of treatingcancer in a subject by determining a level of fatty acid in a samplefrom a subject, and administering an effective amount of a compound offormula II, its pharmaceutically acceptable salts or prodrugs thereof,to the subject wherein the administration is based on the determinationof the level of fatty acid. The compound of formula II, itspharmaceutically acceptable salts or prodrugs thereof inhibit fatty acidsynthesis, thereby treating cancer in the subject.

Typical salts are those of the inorganic ions, such as, for example,sodium, potassium, calcium, magnesium ions, and the like. Such saltsinclude salts with inorganic or organic acids, such as hydrochloricacid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid,succinic acid, lactic acid, mandelic acid, malic acid, citric acid,tartaric acid or maleic acid. In addition, if the compound(s) contain acarboxy group or other acidic group, it can be converted into apharmaceutically acceptable addition salt with inorganic or organicbases. Examples of suitable bases include sodium hydroxide, potassiumhydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,diethanolamine, triethanolamine, and the like.

The PARP inhibitors described herein can contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The PARP inhibitors described herein can also berepresented in multiple tautomeric forms, all of which are includedherein. The PARP inhibitors can also occur in cis- or trans- or E- orZ-double bond isomeric forms. All such isomeric forms of such inhibitorsare expressly included in the present invention. All crystal forms ofthe PARP inhibitors described herein are expressly included in thepresent invention. The PARP inhibitors can also be present as theirpharmaceutically acceptable salts, derivatives or prodrugs.

There are other PARP inhibitors known in the art and they are within thescope of the present invention. The PARP inhibitors have been designedas analogs of benzamides, which bind competitively with the naturalsubstrate NAD in the catalytic site of PARP. The PARP inhibitorsinclude, but are not limited to, benzamides, quinolones andisoquinolones, benzopyrones, methyl3,5-diiodo-4-(4′-methoxyphenoxy)benzoate, and3,5-diiodo-4-(4′-methoxyphenoxy)acetophenone (U.S. Pat. No. 5,464,871,U.S. Pat. No. 5,670,518, U.S. Pat. No. 6,004,978, U.S. Pat. No.6,169,104, U.S. Pat. No. 5,922,775, U.S. Pat. No. 6,017,958, U.S. Pat.No. 5,736,576, and U.S. Pat. No. 5,484,951, all incorporated herein intheir entirety). The PARP inhibitors include a variety of cyclicbenzamide analogs (i.e. lactams) which are potent inhibitors at the NADsite. Other PARP inhibitors include, but are not limited to,benzimidazoles and indoles (EP 841924, EP 1127052, U.S. Pat. No.6,100,283, U.S. Pat. No. 6,310,082, US 2002/156050, US 2005/054631, WO05/012305, WO 99/11628, and US 2002/028815). Other PARP inhibitors knownin the art can also inhibit the synthesis of the fatty acid and arewithin the scope of the present invention (U.S. Application No.60/804,563, filed on Jun. 12, 2006, incorporated herein by reference inits entirety).

Cancer Types

The cancer in the present invention includes but is not limited to,colon adenocarcinoma, esophagus adenocarcinoma, liver hepatocellularcarcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet celltumor, rectum adenocarcinoma, gastrointestinal stromal tumor, stomachadenocarcinoma, adrenal cortical carcinoma, follicular carcinoma,papillary carcinoma, breast cancer, ductal carcinoma, lobular carcinoma,intraductal carcinoma, mucinous carcinoma, phyllodes tumor, ovarianadenocarcinoma, endometrium adenocarcinoma, granulose cell tumor,mucinous cystadenocarcinoma, cervix adenocarcinoma, vulva squamous cellcarcinoma, basal cell carcinoma, prostate adenocarcinoma, giant celltumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma,kidney carcinoma, urinary bladder carcinoma, Wilm's tumor, and lymphoma.

The other examples of the cancer include but are not limited to, adrenalcortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladdercancer, bone cancer, bone metastasis, Adult CNS brain tumors, ChildrenCNS brain tumors, breast cancer, Castleman Disease, cervical cancer,Childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrialcancer, esophagus cancer, Ewing's family of tumors, eye cancer,gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinalstromal tumors, gestational trophoblastic disease, Hodgkin's disease,Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer,acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia,chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer,lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer,malignant mesothelioma, multiple myeloma, myelodysplastic syndrome,nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma,oral cavity and oropharyngeal cancer, osteosarcoma, pancreatic cancer,penile cancer, pituitary tumor, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissuecancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer,testicular cancer, thymus cancer, thyroid cancer, uterine sacrcoma,vaginal cancer, vulvar cancer, and Waldenstr.o:m's macroglobulinemia.

The methods provided by the invention can comprise the administration ofthe PARP inhibitors in combination with other therapies. The choice oftherapy that can be co-administered with the compositions of theinvention can depend, in part, on the condition being treated. Forexample, for treating acute myleoid leukemia, a PARP inhibitor can beused in combination with radiation therapy, monoclonal antibody therapy,chemotherapy, bone marrow transplantation, gene therapy, immunotherapy,or a combination thereof.

Her-2 Related Cancer

In one aspect, the invention provides a method of treating Her-2 relatedcancer by administering an effective amount of PARP inhibitor. Her-2disease is a type of breast cancer. Characterized by aggressive growthand a poor prognosis, it can be caused by the presence of excessivenumbers of a gene called HER2 (human epidermal growth factor receptor-2)in tumor cells. Therapies that can used in combination with the PARPinhibitors as disclosed herein include, but are no limited to Her-2antibodies such as herceptin, anti-hormones (e.g., selective estrogenreceptor modulator (SERM) tamoxifen), chemotherapy and radiotherapy,aromatase inhibitors (e.g. anastrozole, letrozole and exemestane) andanti-estrogens (e.g., fulvestrant (Faslodex)).

Breast Cancer

In one aspect, the invention provides a method of treating breast cancerincluding but not limited to, a ductal carcinoma in duct tissue in amammary gland.

A lobular carcinoma in situ and a ductal carcinoma in situ are breastcancers that develop in the lobules and ducts, respectively, but may nothave spread to the fatty tissue surrounding the breast or to other areasof the body. An infiltrating (or invasive) lobular and a ductalcarcinoma are cancers that have developed in the lobules and ducts,respectively, and have spread to either the breast's fatty tissue and/orother parts of the body. Other cancers of the breast that can benefitfrom treatment provided by the methods of the present invention aremedullary carcinomas, colloid carcinomas, tubular carcinomas, andinflammatory breast cancer.

In some embodiments, the invention provides for treatment of so-called“triple negative” breast cancer. There are several subclasses of breastcancer identified by classic biomarkers such as estrogen receptor (ER)and/or progesterone receptor (PR) positive tumors, HER2-amplifiedtumors, and ER/PR/HER2-negative tumors. These three subtypes have beenreproducibly identified by gene expression profiling in multiple breastcancer and exhibit basal-like subtype expression profiles and poorprognosis. Triple negative breast cancer is characterized byER/PR/HER2-negative tumors.

Ovarian Cancer

In another aspect, the invention provides a method of treating ovariancancer including but not limited to, epithelial ovarian tumors,adenocarcinoma in the ovary and an adenocarcinoma that has migrated fromthe ovary into the abdominal cavity. Treatments for ovarian cancer thatcan be used in combination with the PARP inhibitors of the presentinvention include but are not limited to, surgery, immunotherapy,chemotherapy, hormone therapy, radiation therapy, or a combinationthereof. Some possible surgical procedures include debulking, and aunilateral or bilateral oophorectomy and/or a unilateral or bilateralsalpigectomy.

Anti-cancer drugs that can be used in the combination therapy includecyclophosphamide, etoposide, altretamine, and ifosfamide. Hormonetherapy with the drug tamoxifen can be used to shrink ovarian tumors.Radiation therapy can be external beam radiation therapy and/orbrachytherapy.

Cervical Cancer

In another aspect, the invention provides a method of treating cervicalcancer including but not limited to, an adenocarcinoma in the cervixepithelial. Two main types of this cancer exist: squamous cell carcinomaand adenocarcinomas. Some cervical cancers have characteristics of bothof these and are called adenosquamous carcinomas or mixed carcinomas.

Prostate Cancer

In one other aspect, the invention provides methods to treat prostatecancer including but not limited to, an adenocarcinoma or anadenocarinoma that has migrated to the bone. Prostate cancer develops inthe prostate organ in men, which surrounds the first part of theurethra.

Pancreatic Cancer

In another aspect, the invention provides methods of treating pancreaticcancer including but not limited to, an epitheliod carcinoma in thepancreatic duct tissue and an adenocarcinoma in a pancreatic duct.

Treatments that can be used in combination with the PARP inhibitors ofthe present invention include but are not limited to, surgery,immunotherapy, radiation therapy, and chemotherapy. Possible surgicaltreatment options include a distal or total pancreatectomy and apancreaticoduodenectomy (Whipple procedure). Radiation therapy can be anoption for pancreatic cancer patients, such as external beam radiationwhere radiation is focused on the tumor by a machine outside the body.Another option is intraoperative electron beam radiation administeredduring an operation.

Bladder Cancer

In another aspect, the invention provides methods of treating bladdercancer including but not limited to, a transitional cell carcinoma inurinary bladder. Bladder cancers are urothelial carcinomas (transitionalcell carcinomas) or tumors in the urothelial cells that line thebladder. The remaining cases of bladder cancer are squamous cellcarcinomas, adenocarcinomas, and small cell cancers. Several subtypes ofurothelial carcinomas exist depending on whether they are noninvasive orinvasive and whether they are papillary, or flat. Noninvasive tumors arein the urothelium, the innermost layer of the bladder, while invasivetumors have spread from the urothelium to deeper layers of the bladder'smain muscle wall. Invasive papillary urothelial carcinomas are slenderfinger-like projections that branch into the hollow center of thebladder and also grow outward into the bladder wall. Non-invasivepapillary urothelial tumors grow towards the center of the bladder.While a non-invasive, flat urothelial tumor (also called a flatcarcinoma in situ) is confined to the layer of cells closest to theinside hollow part of the bladder, an invasive flat urothelial carcinomainvades the deeper layer of the bladder, particularly the muscle layer.

The therapies that can be used in combination with the PARP inhibitorsof the present invention for the treatment of bladder cancer includesurgery, radiation therapy, immunotherapy, chemotherapy, or acombination thereof. Some surgical options are a transurethralresection, a cystectomy, or a radical cystectomy. Radiation therapy forbladder cancer can include external beam radiation and brachytherapy.

Immunotherapy is another method that can be used to treat a bladdercancer patient. One method is Bacillus Calmette-Guerin (BCG) where abacterium sometimes used in tuberculosis vaccination is given directlyto the bladder through a catheter. The body mounts an immune response tothe bacterium, thereby attacking and killing the cancer cells. Anothermethod of immunotherapy is the administration of interferons,glycoproteins that modulate the immune response. Interferon alpha isoften used to treat bladder cancer.

Anti-cancer drugs that can be used in combination to treat bladdercancer include thitepa, methotrexate, vinblastine, doxorubicin,cyclophosphamide, paclitaxel, carboplatin, cisplatin, ifosfamide,gemcitabine, or combinations thereof.

Acute Myeloid Leukemia

In another aspect, the invention provides methods of treating acutemyeloid leukemia (AML), preferably acute promyelocytic leukemia inperipheral blood. AML begins in the bone marrow but can spread to otherparts of the body including the lymph nodes, liver, spleen, centralnervous system, and testes. AML is characterized by immature bone marrowcells usually granulocytes or monocytes, which can continue to reproduceand accumulate.

AML can be treated by other therapies in combination with the PARPinhibitors of the present invention. Such therapies include but are notlimited to, immunotherapy, radiation therapy, chemotherapy, bone marrowor peripheral blood stem cell transplantation, or a combination thereof.Radiation therapy includes external beam radiation and can have sideeffects. Anti-cancer drugs that can be used in chemotherapy to treat AMLinclude cytarabine, anthracycline, anthracenedione, idarubicin,daunorubicin, idarubicin, mitoxantrone, thioguanine, vincristine,prednisone, etoposide, or a combination thereof.

Monoclonal antibody therapy can be used to treat AML patients. Smallmolecules or radioactive chemicals can be attached to these antibodiesbefore administration to a patient in order to provide a means ofkilling leukemia cells in the body. The monoclonal antibody, gemtuzumabozogamicin, which binds CD33 on AML cells, can be used to treat AMLpatients unable to tolerate prior chemotherapy regimens. Bone marrow orperipheral blood stem cell transplantation can be used to treat AMLpatients. Some possible transplantation procedures are an allogenic oran autologous transplant.

Other types of leukemia's that can be treated by the methods provided bythe invention include but not limited to, Acute Lymphocytic Leukemia,Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Chronic MyeloidLeukemia, Hairy Cell Leukemia, Myelodysplasia, and MyeloproliferativeDisorders.

Lung Cancer

In another aspect, the invention provides methods to treat lung cancer.The common type of lung cancer is non-small cell lung cancer (NSCLC),which is divided into squamous cell carcinomas, adenocarcinomas, andlarge cell undifferentiated carcinomas. Treatment options for lungcancer in combination with the PARP inhibitors of the present inventioninclude surgery, immunotherapy, radiation therapy, chemotherapy,photodynamic therapy, or a combination thereof. Some possible surgicaloptions for treatment of lung cancer are a segmental or wedge resection,a lobectomy, or a pneumonectomy. Radiation therapy can be external beamradiation therapy or brachytherapy.

Some anti-cancer drugs that can be used in chemotherapy to treat lungcancer include cisplatin, carboplatin, paclitaxel, docetaxel,gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, erlotinib,gefitinib, ifosfamide, methotrexate, or a combination thereof.Photodynamic therapy (PDT) can be used to treat lung cancer patients.

Skin Cancer

In another aspect, the invention provides methods to treat skin cancer.There are several types of cancer that start in the skin. The mostcommon types are basal cell carcinoma and squamous cell carcinoma, whichare non-melanoma skin cancers. Actinic keratosis is a skin conditionthat sometimes develops into squamous cell carcinoma. Non-melanoma skincancers rarely spread to other parts of the body. Melanoma, the rarestform of skin cancer, is more likely to invade nearby tissues and spreadto other parts of the body.

Different types of treatments that can be used in combination with thePARP inhibitors of the present invention include but are not limited to,surgery, radiation therapy, chemotherapy and photodynamic therapy. Somepossible surgical options for treatment of skin cancer are Mohsmicrographic surgery, simple excision, electrodesiccation and curettage,cryosurgery, laser surgery. Radiation therapy can be external beamradiation therapy or brachytherapy. Other types of treatments includebiologic therapy or immunotherapy, chemoimmunotherapy, topicalchemotherapy with fluorouracil and photodynamic therapy.

Eye Cancer, Retinoblastoma

In another aspect, the invention provides methods to treat eyeretinoblastoma. Retinoblastoma is a malignant tumor of the retina. Thetumor can be in one eye only or in both eyes. Treatment options that canbe used in combination with the PARP inhibitors of the present inventioninclude enucleation (surgery to remove the eye), radiation therapy,cryotherapy, photocoagulation, immunotherapy, thermotherapy andchemotherapy. Radiation therapy can be external beam radiation therapyor brachytherapy.

Eye Cancer, Intraocular Melanoma

In another aspect, the invention provides methods to treat intraocular(eye) melanoma. Intraocular melanoma is a disease in which cancer cellsare found in the part of the eye called the uvea. The uvea includes theiris, the ciliary body, and the choroid. Intraocular melanoma occursmost often in people who are middle aged. Treatments that can be used incombination with the PARP inhibitors of the present invention includesurgery, immunotherapy, radiation therapy and laser therapy. Surgery isthe most common treatment of intraocular melanoma. Some possiblesurgical options are iridectomy, iridotrabeculectomy, iridocyclectomy,choroidectomy, enucleation and orbital exenteration. Radiation therapycan be external beam radiation therapy or brachytherapy. Laser therapycan be an intensely powerful beam of light to destroy the tumor,thermotherapy or photocoagulation.

Endometrium Cancer

In another aspect, the invention provides methods to treat endometriumcancer. Endometrial cancer is a cancer that starts in the endometrium,the inner lining of the uterus. Some of the examples of the cancer ofuterus and endometrium include, but are not limited to, adenocarcinomas,adenoacanthomas, adenosquamous carcinomas, papillary serousadenocarcinomas, clear cell adenocarcinomas, uterine sarcomas, stromalsarcomas, malignant mixed mesodermal tumors, and leiomyosarcomas.

Liver Cancer

In another aspect, the invention provides methods to treat primary livercancer (cancer that begins in the liver). Primary liver cancer can occurin both adults and children. Different types of treatments that can beused in combination with the PARP inhibitors of the present inventioninclude surgery, immunotherapy, radiation therapy, chemotherapy andpercutaneous ethanol injection. The types of surgery that can be usedare cryosurgery, partial hepatectomy, total hepatectomy andradiofrequency ablation. Radiation therapy can be external beamradiation therapy, brachytherapy, radiosensitizers or radiolabelantibodies. Other types of treatment include hyperthermia therapy andimmunotherapy.

Kidney Cancer

In another aspect, the invention provides methods to treat kidneycancer. Kidney cancer (also called renal cell cancer or renaladenocarcinoma) is a disease in which malignant cells are found in thelining of tubules in the kidney. Treatments that can be used incombination with the PARP inhibitors of the present invention includesurgery, radiation therapy, chemotherapy and immunotherapy. Somepossible surgical options to treat kidney cancer are partialnephrectomy, simple nephrectomy and radical nephrectomy. Radiationtherapy can be external beam radiation therapy or brachytherapy. Stemcell transplant can be used to treat kidney cancer.

Thyroid Cancer

In another aspect, the invention provides methods to treat thyroidcancer. Thyroid cancer is a disease in which cancer (malignant) cellsare found in the tissues of the thyroid gland. The four main types ofthyroid cancer are papillary, follicular, medullary and anaplastic.Thyroid cancer can be treated by surgery, immunotherapy, radiationtherapy, hormone therapy and chemotherapy. Some possible surgicaloptions that can be used in combination with the PARP inhibitors of thepresent invention include but are not limited to, lobectomy, near-totalthyroidectomy, total thyroidectomy and lymph node dissection. Radiationtherapy can be external radiation therapy or can require intake of aliquid that contains radioactive iodine. Hormone therapy uses hormonesto stop cancer cells from growing. In treating thyroid cancer, hormonescan be used to stop the body from making other hormones that might makecancer cells grow.

AIDS Related Cancers

AIDS-Related Lymphoma

In another aspect, the invention provides methods to treat AIDS-relatedlymphoma. AIDS-related lymphoma is a disease in which malignant cellsform in the lymph system of patients who have acquired immunodeficiencysyndrome (AIDS). AIDS is caused by the human immunodeficiency virus(HIV), which attacks and weakens the body's immune system. The immunesystem is then unable to fight infection and diseases that invade thebody. People with HIV disease have an increased risk of developinginfections, lymphoma, and other types of cancer. Lymphomas are cancersthat affect the white blood cells of the lymph system. Lymphomas aredivided into two general types: Hodgkin's lymphoma and non-Hodgkin'slymphoma. Both Hodgkin's lymphoma and non-Hodgkin's lymphoma can occurin AIDS patients, but non-Hodgkin's lymphoma is more common. When aperson with AIDS has non-Hodgkin's lymphoma, it is called anAIDS-related lymphoma. Non-Hodgkin's lymphomas can be indolent(slow-growing) or aggressive (fast-growing). AIDS-related lymphoma isusually aggressive. The three main types of AIDS-related lymphoma arediffuse large B-cell lymphoma, B-cell immunoblastic lymphoma and smallnon-cleaved cell lymphoma.

Highly-active antiretroviral therapy (HAART) is used to slow progressionof HIV. Medicine to prevent and treat infections, which can be serious,is also used. AIDS-related lymphomas can be treated by chemotherapy,immunotherapy, radiation therapy and high-dose chemotherapy with stemcell transplant. Radiation therapy can be external beam radiationtherapy or brachytherapy. AIDS-related lymphomas can be treated bymonoclonal antibody therapy.

Kaposi's Sarcoma

In one aspect, the invention provides methods to treat Kaposi's sarcoma.Kaposi's sarcoma is a disease in which cancer cells are found in thetissues under the skin or mucous membranes that line the mouth, nose,and anus. Kaposi's sarcoma can occur in people who are takingimmunosuppressants. Kaposi's sarcoma in patients who have AcquiredImmunodeficiency Syndrome (AIDS) is called epidemic Kaposi's sarcoma.Kaposi's sarcoma can be treated with surgery, chemotherapy, radiationtherapy and immunotherapy. External radiation therapy is a commontreatment of Kaposi's sarcoma. Treatments that can be used incombination with the PARP inhibitors of the present invention includebut are not limited to, local excision, electrodeiccation and curettage,and cryotherapy.

Viral-Induced Cancers

In another aspect, the invention provides methods to treat viral-inducedcancers. The major virus-malignancy systems include hepatitis B virus(HBV), hepatitis C virus (HCV), and hepatocellular carcinoma; humanlymphotropic virus-type 1 (HTLV-1) and adult T-cell leukemia/lymphoma;and human papilloma virus (HPV) and cervical cancer.

Virus-Induced Hepatocellular Carcinoma

HBV and HCV and hepatocellular carcinoma or liver cancer can appear toact via chronic replication in the liver by causing cell death andsubsequent regeneration. Treatments that can be used in combination withthe PARP inhibitors of the present invention include but are not limitedto, include surgery, immunotherapy, radiation therapy, chemotherapy andpercutaneous ethanol injection. The types of surgery that can be usedare cryosurgery, partial hepatectomy, total hepatectomy andradiofrequency ablation. Radiation therapy can be external beamradiation therapy, brachytherapy, radiosensitizers or radiolabelantibodies. Other types of treatment include hyperthermia therapy andimmunotherapy.

Viral-Induced Adult T Cell Leukemia/Lymphoma

Adult T cell leukemia is a cancer of the blood and bone marrow. Thetreatments for adult T cell leukemia/lymphoma that can be used incombination with the PARP inhibitors of the present invention includebut are not limited to, radiation therapy, immunotherapy, andchemotherapy. Radiation therapy can be external beam radiation therapyor brachytherapy. Other methods of treating adult T cellleukemia/lymphoma include immunotherapy and high-dose chemotherapy withstem cell transplantion.

Viral-Induced Cervical Cancer

Infection of the cervix with human papillomavirus (HPV) is a cause ofcervical cancer. The treatments for cervical cancers that can be used incombination with the PARP inhibitors of the present invention includebut are not limited to, surgery, immunotherapy, radiation therapy andchemotherapy. The types of surgery that can be used are conization,total hysterectomy, bilateral salpingo-oophorectomy, radicalhysterectomy, pelvic exenteration, cryosurgery, laser surgery and loopelectrosurgical excision procedure. Radiation therapy can be externalbeam radiation therapy or brachytherapy.

CNS Cancers

Brain and spinal cord tumors are abnormal growths of tissue found insidethe skull or the bony spinal column, which are the primary components ofthe central nervous system (CNS). Benign tumors are noncancerous, andmalignant tumors are cancerous. Tumors that originate in the brain orspinal cord are called primary tumors. Primary tumors can result fromspecific genetic disease (e.g., neurofibromatosis, tuberous sclerosis)or from exposure to radiation or cancer-causing chemicals.

The primary brain tumor in adults comes from cells in the brain calledastrocytes that make up the blood-brain barrier and contribute to thenutrition of the central nervous system. These tumors are called gliomas(astrocytoma, anaplastic astrocytoma, or glioblastoma multiforme). Someof the tumors are, but not limited to, Oligodendroglioma, Ependymoma,Meningioma, Lymphoma, Schwannoma, and Medulloblastoma.

Neuroepithelial Tumors of the CNS

Astrocytic tumors, such as astrocytoma; anaplastic (malignant)astrocytoma, such as hemispheric, diencephalic, optic, brain stem,cerebellar; glioblastoma multiforme; pilocytic astrocytoma, such ashemispheric, diencephalic, optic, brain stem, cerebellar; subependymalgiant cell astrocytoma; and pleomorphic xanthoastrocytoma.Oligodendroglial tumors, such as oligodendroglioma; and anaplastic(malignant) oligodendroglioma. Ependymal cell tumors, such asependymoma; anaplastic ependymoma; myxopapillary ependymoma; andsubependymoma. Mixed gliomas, such as mixed oligoastrocytoma; anaplastic(malignant) oligoastrocytoma; and others (e.g. ependymo-astrocytomas).Neuroepithelial tumors of uncertain origin, such as polarspongioblastoma; astroblastoma; and gliomatosis cerebri. Tumors of thechoroid plexus, such as choroid plexus papilloma; and choroid plexuscarcinoma (anaplastic choroid plexus papilloma). Neuronal and mixedneuronal-glial tumors, such as gangliocytoma; dysplastic gangliocytomaof cerebellum (Lhermitte-Duclos); ganglioglioma; anaplastic (malignant)ganglioglioma; desmoplastic infantile ganglioglioma, such asdesmoplastic infantile astrocytoma; central neurocytoma;dysembryoplastic neuroepithelial tumor; olfactory neuroblastoma(esthesioneuroblastoma. Pineal Parenchyma Tumors, such as pineocytoma;pineoblastoma; and mixed pineocytoma/pineoblastoma. Tumors withneuroblastic or glioblastic elements (embryonal tumors), such asmedulloepithelioma; primitive neuroectodermal tumors with multipotentdifferentiation, such as medulloblastoma; cerebral primitiveneuroectodermal tumor; neuroblastoma; retinoblastoma; andependymoblastoma.

Other CNS Neoplasms

Tumors of the sellar region, such as pituitary adenoma; pituitarycarcinoma; and craniopharyngioma. Hematopoietic tumors, such as primarymalignant lymphomas; plasmacytoma; and granulocytic sarcoma. Germ CellTumors, such as germinoma; embryonal carcinoma; yolk sac tumor(endodermal sinus tumor); choriocarcinoma; teratoma; and mixed germ celltumors. Tumors of the Meninges, such as meningioma; atypical meningioma;and anaplastic (malignant) meningioma. Non-menigothelial tumors of themeninges, such as Benign Mesenchymal; Malignant Mesenchymal; PrimaryMelanocytic Lesions; Hemopoietic Neoplasms; and Tumors of UncertainHistogenesis, such as hemangioblastoma (capillary hemangioblastoma).Tumors of Cranial and Spinal Nerves, such as schwannoma (neurinoma,neurilemoma); neurofibroma; malignant peripheral nerve sheath tumor(malignant schwannoma), such as epithelioid, divergent mesenchymal orepithelial differentiation, and melanotic. Local Extensions fromRegional Tumors; such as paraganglioma (chemodectoma); chordoma;chodroma; chondrosarcoma; and carcinoma. Metastatic tumours,Unclassified Tumors and Cysts and Tumor-like Lesions, such as Rathkecleft cyst; Epidermoid; dermoid; colloid cyst of the third ventricle;enterogenous cyst; neuroglial cyst; granular cell tumor (choristoma,pituicytoma); hypothalamic neuronal hamartoma; nasal glial herterotopia;and plasma cell granuloma.

Chemotherapeutics available are, but not limited to, alkylating agentssuch as, Cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU,CCNU, Decarbazine, Procarbazine, Busulfan, and Thiotepa; antimetabolitessuch as, Methotraxate, 5-Fluorouracil, Cytarabine, Gemcitabine(Gemzar®), 6-mercaptopurine, 6-thioguanine, Fludarabine, and Cladribine;anthracyclins such as, daunorubicin. Doxorubicin, Idarubicin, Epirubicinand Mitoxantrone; antibiotics such as, Bleomycin; camptothecins such as,irinotecan and topotecan; taxanes such as, paclitaxel and docetaxel; andplatinums such as, Cisplatin, carboplatin, and Oxaliplatin.

PNS Cancers

The peripheral nervous system consists of the nerves that branch outfrom the brain and spinal cord. These nerves form the communicationnetwork between the CNS and the body parts. The peripheral nervoussystem is further subdivided into the somatic nervous system and theautonomic nervous system. The somatic nervous system consists of nervesthat go to the skin and muscles and is involved in conscious activities.The autonomic nervous system consists of nerves that connect the CNS tothe visceral organs such as the heart, stomach, and intestines. Itmediates unconscious activities.

Acoustic neuromas are benign fibrous growths that arise from the balancenerve, also called the eighth cranial nerve or vestibulocochlear nerve.The malignant peripheral nerve sheath tumor (MPNST) is the malignantcounterpart to benign soft tissue tumors such as neurofibromas andschwannomas. It is most common in the deep soft tissue, usually in closeproximity of a nerve trunk. The most common sites include the sciaticnerve, brachial plexus, and sarcal plexus.

The MPNST can be classified into three major categories withepithelioid, mesenchymal or glandular characteristics. Some of the MPNSTinclude but not limited to, subcutaneous malignant epithelioidschwannoma with cartilaginous differentiation, glandular malignantschwannoma, malignant peripheral nerve sheath tumor with perineurialdifferentiation, cutaneous epithelioid malignant nerve sheath tumor withrhabdoid features, superficial epithelioid MPNST, triton Tumor (MPNSTwith rhabdomyoblastic differentiation), schwannoma with rhabdomyoblasticdifferentiation. Rare MPNST cases contain multiple sarcomatous tissuetypes, especially osteosarcoma, chondrosarcoma and angiosarcoma. Thesehave sometimes been indistinguishable from the malignant mesenchymoma ofsoft tissue.

Other types of PNS cancers include but not limited to, malignant fibrouscytoma, malignant fibrous histiocytoma, malignant meningioma, malignantmesothelioma, and malignant mixed Müllerian tumor.

Oral Cavity and Oropharyngeal Cancer

Cancers of the oral cavity include but are not limited to,hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, andoropharyngeal cancer.

Stomach Cancer

There are three main types of stomach cancers: lymphomas, gastricstromal tumors, and carcinoid tumors. Lymphomas are cancers of theimmune system tissue that are sometimes found in the wall of thestomach. Gastric stromal tumors develop from the tissue of the stomachwall. Carcinoid tumors are tumors of hormone-producing cells of thestomach.

Testicular Cancer

Testicular cancer is cancer that typically develops in one or bothtesticles in young men. Cancers of the testicle develop in certain cellsknown as germ cells. The two types of germ cell tumors (GCTs) that occurin men are seminomas (60%) and nonseminomas (40%). Tumors can also arisein the supportive and hormone-producing tissues, or stroma, of thetesticles. Such tumors are known as gonadal stromal tumors. The twotypes are Leydig cell tumors and Sertoli cell tumors. Secondarytesticular tumors are those that start in another organ and then spreadto the testicle. Lymphoma is a secondary testicular cancer.

Thymus Cancer

The thymus is a small organ located in the upper/front portion of yourchest, extending from the base of the throat to the front of the heart.The thymus contains two main types of cells, thymic epithelial cells andlymphocytes. Thymic epithelial cells can give origin to thymomas andthymic carcinomas. Lymphocytes, whether in the thymus or in the lymphnodes, can become malignant and develop into cancers called Hodgkindisease and non-Hodgkin lymphomas. The thymus cancer includes Kulchitskycells, or neuroendocrine cells, which normally release certain hormones.These cells can give rise to cancers, called carcinoids or carcinoidtumors.

Treatments that can be used in combination with the PARP inhibitors ofthe present invention include but are not limited to, surgery,immunotherapy, chemotherapy, radiation therapy, combination ofchemotherapy and radiation therapy or biological therapy. Anticancerdrugs that have been used in the treatment of thymomas and thymiccarcinomas are doxorubicin (Adriamycin), cisplatin, ifosfamide, andcorticosteroids (prednisone).

Nutritional and Metabolic Disorders

The examples of nutritional and metabolic disorders include, but are notlimited to, diabetes insipidus, fabry, fatty acid metabolism disorders,galactosemia, gaucher, glucose-6-phosphate dehydrogenase (G6PD),glutaric aciduria, hurler, hurler-scheie, hunter, hypophosphatemia,1-cell, krabbe, lactic acidosis, long chain 3 hydroxyacyl CoAdehydrogenase deficiency (LCHAD), lysosomal storage diseases,mannosidosis, maple syrup urine, maroteaux-lamy, metachromaticleukodystrophy, mitochondrial, morquio, mucopolysaccharidosis,neuro-metabolic, niemann-pick, organic acidemias, purine,phenylketonuria (PKU), pompe, pseudo-hurler, pyruvate dehydrogenasedeficiency, sandhoff, sanfilippo, scheie, sly, tay-sachs,trimethylaminuria (fish-malodor syndrome), urea cycle conditions,vitamin D deficiency rickets, metabolic disease of muscle, inheritedmetabolic disorders, acid-base imbalance, acidosis, alkalosis,alkaptonuria, alpha-mannosidosis, amyloidosis, anemia, iron-deficiency,ascorbic acid deficiency, avitaminosis, beriberi, biotinidasedeficiency, deficient glycoprotein syndrome, carnitine disorders,cystinosis, cystinuria, fabry disease, fatty acid oxidation disorders,fucosidosis, galactosemias, gaucher disease, gilbert disease,glucosephosphate dehydrogenase deficiency, glutaric academia, glycogenstorage disease, hartnup disease, hemochromatosis, hemosiderosis,hepatolenticular degeneration, histidinemia, homocystinuria,hyperbilirubinemia, hypercalcemia, hyperinsulinism, hyperkalemia,hyperlipidemia, hyperoxaluria, hypervitaminosis A, hypocalcemia,hypoglycemia, hypokalemia, hyponatremia, hypophosphotasia, insulinresistance, iodine deficiency, iron overload, jaundice, chronicidiopathic, leigh disease, Lesch-Nyhan syndrome, leucine metabolismdisorders, lysosomal storage diseases, magnesium deficiency, maple syrupurine disease, MELAS syndrome, menkes kinky hair syndrome, metabolicsyndrome X, mucolipidosis, mucopolysacchabridosis, Niemann-Pick disease,obesity, ornithine carbamoyltransferase deficiency disease,osteomalacia, pellagra, peroxisomal disorders, porphyria,erythropoietic, porphyries, progeria, pseudo-gaucher disease, refsumdisease, reye syndrome, rickets, sandhoff disease, tangier disease,Tay-sachs disease, tetrahydrobiopterin deficiency, trimethylaminuria(fish odor syndrome), tyrosinemias, urea cycle disorders,water-electrolyte imbalance, wernicke encephalopathy, vitamin Adeficiency, vitamin B12 deficiency, vitamin B deficiency, wolmandisease, and zellweger syndrome.

In some preferred embodiments, the metabolic diseases include diabetesand obesity.

Methods of Use

The compounds suitable for use in the present invention are compoundsthat inhibit fatty acid synthesis. Preferably the inhibitors are PARPinhibitors. The analysis of the level of fatty acid before and/or aftertreatment with an effective amount of PARP inhibitors has varioustherapeutic and diagnostic applications. Clinical applications include,for example, detection of disease, distinguishing disease states toinform prognosis, selection of therapy such as, treatment with aneffective amount of PARP inhibitors, prediction of therapeutic response,disease staging, identification of disease processes, prediction ofefficacy of therapy with PARP inhibitors, monitoring of patientstrajectories (e.g., prior to onset of disease), prediction of adverseresponse to PARP inhibitors, monitoring of therapy associated efficacyand toxicity, and detection of recurrence. An identification of thelevel of the fatty acid in a subject can also be used to select atherapy and a personalized dose regimen for a subject for treatment witha PARP inhibitor.

The identification of the level of fatty acid in a subject andsubsequent treatment with an effective amount of PARP inhibitors can beused to enable or assist in the pharmaceutical drug development processfor therapeutic agents. The identification of the level of fatty acidcan be used to select subjects enrolling in a clinical trial for PARPinhibitors. Further identification of the level of fatty acid canindicate the state of the disease of subjects undergoing treatment inclinical trials, and show changes in the state during the treatment withPARP inhibitors. The identification of the level of fatty acid candemonstrate the efficacy of treatment with PARP inhibitors, and can beused to stratify subjects according to their responses to varioustherapies. The identification of the level of the fatty acid can also beused to select a personalized dose regimen for the subject for treatmentwith PARP inhibitors.

In certain embodiments, patients, health care providers, such as doctorsand nurses, or health care managers, select a treatment of a subjectwith an effective amount of PARP inhibitors based on the level of thefatty acid in a sample from the subject. The methods can be used toevaluate the efficacy of treatments over time. For example, biologicalsamples can be obtained from a patient over a period of time as thepatient is undergoing treatment with PARP inhibitors. The level of fattyacid in the different samples can be compared to each other to determinethe efficacy of the treatment. Also, the methods described herein can beused to compare the efficacies of different disease therapies includingtreatment with PARP inhibitors, and/or responses to one or moretreatments in different populations (e.g., ethnicities, familyhistories, etc.).

Formulation and Pharmaceutical Compositions

The methods provided by the invention can comprise the administration ofan effective amount of inhibitors as provided herein, in combinationwith other therapies. The choice of therapy that can be co-administeredwith the compositions of the invention will depend, in part, on thecondition being treated. For example, for treating acute myeloidleukemia, compound of some embodiments of the invention can be used incombination with radiation therapy, monoclonal antibody therapy,chemotherapy, bone marrow transplantation, or a combination thereof.

Radiosensitizers can be administered in conjunction with atherapeutically effective amount of PARP inhibitors, where the PARPinhibitors can promote the incorporation of radiosensitizers to thetarget cells or the PARP inhibitors can control the flow oftherapeutics, nutrients, and/or oxygen to the target cells. Similarly,chemosensitizers are also known to increase the sensitivity of cancerouscells to the toxic effects of chemotherapeutic compounds. Exemplarychemotherapeutic agents that can be used in conjunction with PARPinhibitors include, but are not limited to, adriamycin, camptothecin,dacarbazine, carboplatin, cisplatin, daunorubicin, docetaxel,doxorubicin, interferon (alpha, beta, gamma), interleukin 2, innotecan,paclitaxel, streptozotocin, temozolomide, topotecan, and therapeuticallyeffective analogs and derivatives of the same. In addition, othertherapeutic agents which can be used in conjunction with a PARPinhibitors include, but are not limited to, 5-fluorouracil, leucovorin,5′-amino-5′-deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol-DA), 2,3-DPG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andL-BSO.

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Many cancertreatment protocols currently employ radiosensitizers activated by theelectromagnetic radiation of x-rays. Examples of x-ray activatedradiosensitizers include, but are not limited to, the following:metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, photofrin, benzoporphyrin derivatives,NPe6, tin etioporphyrin SnET2, pheoborbide, bacteriochlorophyll,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

The methods of treatment as disclosed herein can be via oraladministration, transmucosal administration, buccal administration,nasal administration, inhalation, parental administration, intravenous,subcutaneous, intramuscular, sublingual, transdermal administration, andrectal administration.

Non-invasive administration includes (1) topical application to the skinin the form of an ointment or cream; (2) direct topical application tooropharyngeal tissues; (3) oral administration; (4) nasal administrationas an aerosol; (5) intravaginal application of the inhibitor in the formof a suppository, cream or foam; (6) direct application to the uterinecervix; (7) rectal administration via suppository, irrigation or othersuitable means; (8) bladder irrigation; and (9) administration ofaerosolized formulation of the inhibitor to the lung.

Pharmaceutical compositions of PARP inhibitors for the methods of thepresent invention include compositions wherein the active ingredient iscontained in a therapeutically or prophylactically effective amount. Theactual amount effective for a particular application will depend, interalia, on the condition being treated and the route of administration.Determination of an effective amount is well within the capabilities ofthose skilled in the art. The pharmaceutical compositions comprise thePARP inhibitors, one or more pharmaceutically acceptable carriers,diluents or excipients, and optionally additional therapeutic agents.The compositions can be formulated for sustained or delayed release.

In some embodiments, the PARP inhibitors can be administered locally ortopically in gels, ointments, solutions, impregnated bandages,liposomes, or biodegradable microcapsules. Compositions or dosage formsfor topical application can include solutions, lotions, ointments,creams, gels, suppositories, sprays, aerosols, suspensions, dustingpowder, impregnated bandages and dressings, liposomes, biodegradablepolymers, and artificial skin. Typical pharmaceutical carriers whichmake up the foregoing compositions include alginates,carboxymethylcellulose, methylcellulose, agarose, pectins, gelatins,collagen, vegetable oils, mineral oils, stearic acid, stearyl alcohol,petrolatum, polyethylene glycol, polysorbate, polylactate,polyglycolate, polyanhydrides, phospholipids, polyvinylpyrrolidone, andthe like.

The compositions can be administered by injection, topically, orally,transdermally, rectally, or via inhalation. The oral form in which thetherapeutic agent is administered can include powder, tablet, capsule,solution, or emulsion. The effective amount can be administered in asingle dose or in a series of doses separated by appropriate timeintervals, such as hours. Pharmaceutical compositions can be formulatedin conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Suitable techniques for preparing pharmaceuticalcompositions of the therapeutic agents of the present invention are wellknown in the art.

It will be appreciated that appropriate dosages of the active compounds,and compositions comprising the active compounds, can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular PARP inhibitor, the routeof administration, the time of administration, the rate of excretion ofthe compound, the duration of the treatment, other drugs, compounds,and/or materials used in combination, and the age, sex, weight,condition, general health, and prior medical history of the patient. Theamount of compound and route of administration will ultimately be at thediscretion of the physician, although generally the dosage will be toachieve local concentrations at the site of action which achieve thedesired effect without causing substantial harmful or deleteriousside-effects.

Administration in vivo can be effected in one dose, continuously orintermittently (e.g. in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician.

EXAMPLES Example I

Goal: This study relates to analyzing the effect of the3-nitro-4-iodobenzamide (compound of formula III) in vitro on culturedOVCAR-3 cells' and HeLa cells' metabolic fluxes using[1,2-¹³C₂]-D-glucose as the only source of glucose. The analysisincludes correlating fluxes with cell growth-modifying effect, analyzingmechanism of anti-proliferative action and analyzing potential toxicity,selectivity, and efficacy. Target metabolites include glucose (culturemedium and pellet glycogen), lactate (culture medium), ¹³CO₂ (culturemedium), C:14 (myristate); C:16 (palmitate); C:18 (stearate); C:18-1(oleate); C:20; C:22; C:24 (cell pellet), acetyl-CoA synthesis for fattyacids (cell pellet), and RNA ribose and DNA deoxyribose (cell pellet).Target fluxes include glucose uptake from culture media; lactateproduction from glucose (anaerobic glycolysis); ¹³CO₂ release fromglucose via TCA cycle; glycogen synthesis; de novo fatty acid synthesis,elongation, desaturation and acetyl-CoA synthesis; and pentose cycle-RNAand DNA ribose synthesis via oxidative and non-oxidative reactions.

Materials & Methods: The tracer for this metabolic profiling study,stable isotope [1,2-¹³C₂]-D-glucose, is purchased with >99% purity and99% isotope enrichment for each position from Cambridge IsotopeLaboratories, Inc. (Andover, Mass.).

Cells & cell culture: OVCAR3 and Hela cells are purchased from AmericanType Culture Collection (ATCC). The cells are cultured according toinstructions obtained from ATCC. The cells are incubated at 37° C., 5%CO₂ and 95% humidity and passed by using trypsin 0.25% (Gibco/BRL) nomore than ten times after receipt from the ATCC and prior to use in thisstudy.

Seventy-five percent confluent cultures cells are incubated in[1,2-¹³C₂]D-glucose-containing media (100 mg/dl total concentration=5mM; 50% isotope enrichment—i.e. half unlabeled glucose, half labeledwith the stable isotope ¹³C tracer). Cells are plated at a density of10⁶ per T75 culture flask and 3-nitro-4-iodobenzamide is added in aconcentration range of 0.3 and 3 μM to culture media. Control culturesare treated with vehicle only. The doses of 3-nitro-4-iodobenzamide forthe present study are selected based on in vitro experimentsdemonstrating that this drug effectively controls glycogen phosphorylaseactivity in the presence or absence of glucose in human cells (AndersenB, et al. 2002, Biochem J. 367: 443-450). Glucose and lactate levels inthe medium are measured using a Cobas Mira chemistry analyzer (RocheDiagnostics, Pleasanton, Calif., USA).

RNA ribose stable isotope studies: RNA ribose is isolated by acidhydrolysis of cellular RNA after Trizol purification of cell extracts.Total RNA amounts are assessed by spectrophotometric determination, intriplicate cultures. Ribose is derivatized to its aldonitrile acetateform using hydroxylamine in pyridine with acetic anhydride (Supelco,Bellefonte, Penn.) before mass spectral analyses. The ion cluster ismonitored around the m/z 256 (carbons 1-5 of ribose) (chemicalionization, CI) and m/z 217 (carbons 3-5 of ribose) and m/z 242 (carbons1-4 of ribose) (electron impact ionization, EI) to determine molarenrichment and the positional distribution of ¹³C in ribose. ByConvention, the base mass of ¹²C-compounds (with their deriviatizationagents) is given as m_(o) as measured by mass spectrometry (Boros L G,et al. 2002, Drug Discov. Today 7: 364-372).

Ribose molecules labeled with a single ¹³C atom on the first carbonposition (m1) recovered from RNA are used to gauge the ribose fractionproduced by direct oxidation of glucose through the G6PD pathway. Ribosemolecules labeled with ¹³C on the first two carbon positions (m2) areused to measure the fraction produced by transketolase. Doubly labeledribose molecules (m₂ and m₄) on the fourth and fifth carbon positionsare used to measure molar fraction produced by triose phosphateisomerase and transketolase.

Lactate: Lactate from the cell culture media (0.2 ml) is extracted byethylene chloride after acidification with HCL. Lactate is derivatizedto its propylamine-heptafluorobutyrate ester form and the m/z 328(carbons 1-3 of lactate) (chemical ionization, CI) is monitored for thedetection of m₁ (recycled lactate through the PC) and m₂ (lactateproduced by the Embden-Meyerhof-Parnas pathway) for the estimation ofpentose cycle activity (Lee W N, et al. 1998, Am J Physiol 274:E843-E851). The m₁/m₂ ratios produced in lactate are recorded andreleased by OVCAR3 and Hela cells in order to determine pentose cycleactivity versus anaerobic glycolysis in response to3-nitro-4-iodobenzamide treatment.

Glutamate: Glutamate label distribution from glucose is suitable fordetermining glucose oxidation versus anabolic glucose use within the TCAcycle, also known as anaplerotic flux. Tissue culture medium is firsttreated with 6% perchloric acid and the supernatant is passed through a3 cm³ Dowex-50 (H⁺) column. Amino acids are eluted with 15 ml 2Nammonium hydroxide. To further separate glutamate from glutamine, theamino acid mixture is passed through a 3 cm³ Dowex-1 (acetate) column,and then collected with 15 ml 0.5 N acetic acid. The glutamate fractionfrom the culture medium is converted to its trifluoroacetyl butyl ester(TAB). Under EI conditions, ionization of TAB-glutamate produces twofragments, m/z 198 and m/z 152, corresponding to C2-C5 and C2-C4 ofglutamate (Lee W N, et al. 1996, Developmental Neuroscience 18:469-477). Glutamate labeled on the 4-5 carbon positions indicatespyruvate dehydrogenase activity while glutamate labeled on the 2-3carbon positions indicates pyruvate carboxylase activity for the entryof glucose carbons to the TCA cycle. TCA cycle anabolic glucoseutilization is calculated based on the m₁/m₂ ratios of glutamate (LeimerK R, et al. 1977, J. Chromatography 141: 121-144).

Fatty acids: Palmitate, stearate, cholesterol and oleate are extractedafter saponification of cell pellets in 30% KOH and 100% ethanol usingpetroleum ether. Fatty acids are converted to their methylatedderivative using 0.5N methanolic-HCL. Palmitate, stearate and oleate aremonitored at m/z 270, m/z 298 and m/z 264, respectively, with theenrichment of ¹³C labeled acetyl units which reflect synthesis,elongation and desaturation of the new lipid fraction as determined bymass isotopomer distribution analysis (MIDA) of different isotopomers(Lee W N, et al. 1998, J. Biol. Chem. 273: 20929-20934; Lee W N, et al.1995, Anal Biochem 226: 100-112).

Gas Chromatography/Mass Spectrometry (GC/MS): Mass spectral data areobtained on the HP5973 mass selective detector connected to an HP6890gas chromatograph. The settings are as follows: GC inlet 250° C.,transfer line 280° C., MS source 230° C., MS Quad 150° C. An HP-5capillary column (30 m length, 250 μm diameter, 0.25 μm film thickness)is used for glucose, ribose and lactate analyses. Transketolase has thehighest metabolic control coefficient in the non-oxidative branch of thepentose cycle (Sabate L, et al. 1995, Mol. Cell. Biochem. 142: 9-17;Comin-Anduix B, et al. 2001, Eur. J. Biochem. 268: 4177-4182). It shouldbe noted, though, that transketolase and transaldolase, besides otherenzymes, all can participate in non-oxidative pentose cycle metabolismin human cells.

Data analysis and statistical methods: Each experiment is carried outusing triplicate cell cultures for each condition within each experimentand experiments are repeated once. Mass spectroscopic analyses iscarried out by three independent automatic injections of 1 μl samples bythe automatic sampler and accepted only if the standard sample deviationis less than 1% of the normalized peak intensity. Statistical analysisis performed using the Student's t-test for unpaired samples. Two-tailedsignificance at the 99% confidence interval (u+/−2.58σ), p<0.01indicates significant differences in glucose carbon metabolism incontrol and 3-nitro-4-iodobenzamide.

Results: Tracer treatment is successful in HeLa cells and all cultures'tracer labeled glucose fraction is between 45% and 55% of total glucoseat 0.0 minutes. Twenty four, forty eight and seventy two hours of tracerincubation is sufficient to generate HeLa cell specific metabolitelabeling profiles. There are differences in HeLa cell glucose metabolismin response to 3-nitro-4-iodobenzamide. The effects are dose-responsiveand are most consistent with decreased medium and long chain fatty acidde novo synthesis from glucose. De novo synthesis of arachidic acid(C:20) is spared from the inhibitory action of 3-nitro-4-iodobenzamide.Acetyl-CoA enrichment from glucose for arachidic acid synthesis is highin the 3-nitro-4-iodobenzamide treated HeLa cultures.

Oxidation of glucose into CO₂ and ATP production in the TCA cycle arenot affected by 3-nitro-4-iodobenzamide. There is a dose-dependentincrease in acetyl-CoA contribution from glucose to long chain saturatedfatty acids (C:20-C:24). 3-nitro-4-iodobenzamide is a metabolicallyactive compound in HeLa cells. De novo medium and long chain fatty acid(C:14-C:18) syntheses are decreased with low contribution to acetyl-CoAsynthesis from glucose. 3-nitro-4-iodobenzamide decreases cell membraneformation via limited de novo fatty acid synthesis as the underlyingmechanism of its anti-proliferative action in HeLa cells.3-nitro-4-iodobenzamide increases acetyl-CoA formation from glucose forarachidic acid formation and therefore it can stimulate prostaglandinsynthesis. 3-nitro-4-iodobenzamide has no toxic effects on cell energyproduction, nucleic acid turnover and glycogen synthesis.3-nitro-4-iodobenzamide has no inhibitory effect on substrate (glucose)uptake and activation (phosphorylation).

Tracer treatment is successful in OVCAR-3 cells and all cultures' tracerlabeled glucose fraction is between 45% and 55% of total glucose at 0.0minutes. Seventy two hours of tracer incubation is sufficient togenerate HeLa cell specific metabolite labeling profiles. There aresignificant differences in OVCAR-3 cell glucose metabolism in responseto 3-nitro-4-iodobenzamide. The effects are dose-responsive and are mostconsistent with decreased medium and long chain fatty acid de novosynthesis from glucose as well as decreased acetyl-CoA synthesis fromglucose.

Medium and long chain fatty acid de novo synthesis (C:14-C:18) arespeared from the inhibitory action of 3-nitro-4-iodobenzamide in OVCAR-3cells. De novo fatty acid synthesis is relatively low (<30%) in OVCAR-3cells. Aldolase and triose phosphate isomerase are active during DNAsynthesis and transketolase as well as G6PDH are target enzymes of3-nitro-4-iodobenzamide treatment. Glycogen synthesis and breakdown viathe direct pathway is inhibited by 3-nitro-4-iodobenzamide.

There is a dose-dependent increase in acetyl-CoA contribution fromglucose to long chain saturated fatty acids (C:20-C:24).3-nitro-4-iodobenzamide is a metabolically active compound in OVCAR-3cells. De novo medium and long chain fatty acid (C:14-C:18) synthesesare slightly increased with no significant change in acetyl-CoAsynthesis from glucose. 3-nitro-4-iodobenzamide decreases cell membraneformation via limited de novo long chain (C:20-C:24) fatty acidsynthesis as the underlying mechanism of its anti-proliferative actionin OVCAR-3 cells. The 3-nitro-4-iodobenzamide also inhibits G6PDH andtransketolase fluxes for DNA deoxyribose synthesis and direct glycogensynthesis. The 3-nitro-4-iodobenzamide has no toxic effects on cellenergy production, and nucleic acid RNA turnover. The results of thesestudies are depicted in FIGS. 3A, 3B (¹³C-labeled myristate (C:14) at 72hours), 4A, 4B ((¹³C-labeled palmitate (C:16) at 72 hours), 5A, 5B(13C-labeled stearate (C:18) at 72 hours), 6A, 6B (¹³C-labeled oleate(C:18-1) at 72 hours), 7A, 7B (¹³C-labeled C:22 fatty acid at 72 hours),8A, 8B (¹³C-labeled C:24 fatty acid at 72 hours), 9A, 9B (¹³C-labeledarachidic acid (C:20) at 72 hours), 10A, 10B (¹³C-labeled C:22 fattyacid at 72 hours), and 11A, 11B (¹³C-labeled C:24 fatty acid at 72hours). In each case, the “A” figure shows the amount of fatty acidincorporation in the cell pellet, while the “B” figure shows the ¹³Acetyl Co-A enrichment from glucose (left) and the fraction of new fattyacid synthesis (right). These results demonstrate that the PARP-1inhibitor 3-nitro-4-iodobenzamide inhibits fatty acid synthesis incells.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of treating a fatty acid synthesis related diseasecomprising administering to a patient in need thereof an effectiveamount of a compound of formula Ia or metabolite thereof or apharmaceutically acceptable salt thereof to inhibit a fatty acidsynthesis, wherein said fatty acid synthesis related disease is obesity,and wherein the compound of formula Ia is a compound of the formula:

wherein R₁, R₂, R₃, R₄, and R₅ are, independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, bromo,fluoro, chloro, (C₁-C₆) alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, andphenyl, wherein at least two of the five R₁, R₂, R₃, R₄, and R₅substituents are always hydrogen, at least one of the five substituentsis always nitro, and at least one substituent positioned adjacent to anitro is always iodo, or a metabolite thereof or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein said fattyacid synthesis inhibited is synthesis of a medium chain fatty acid or along chain fatty acid.
 3. The method of claim 1, wherein said inhibitionof said fatty acid synthesis comprises inhibiting at least one enzyme ofa glucose pathway.
 4. The method of claim 1, wherein said inhibition ofsaid fatty acid synthesis comprises inhibiting at least one enzyme of afatty acid biosynthetic pathway.
 5. The method of claim 1, wherein saidinhibition of said fatty acid synthesis comprises inhibiting at leastone enzyme selected from the group consisting of acetyl Co-A, malonylCo-A, acetyl Co-A carboxylase, and fatty acid synthase.
 6. The method ofclaim 1, wherein said inhibition of said fatty acid synthesis comprisesinhibiting at least one enzyme of a fatty acid synthase.
 7. The methodof claim 6, wherein said fatty acid synthase comprises acyl carrierprotein, acetyl transferase, malonyl transferase, 3-keto-acyl-ACPsynthase, 3-ketoacyl-ACP reductase, 3-hydroxy-acyl-ACP dehydratase, andenoyl-ACP reductase.
 8. The method of claim 1, wherein said inhibitionof said fatty acid synthesis comprises inhibiting synthesis of anacetyl-CoA from a glucose.
 9. The method of claim 1, wherein saidinhibition of said fatty acid synthesis comprises inhibiting said fattyacid synthesis from an acetyl-CoA.
 10. The method of claim 1, whereinsaid inhibition is determined by analyzing a metabolite or a molecularflux of a glucose pathway or a fatty acid biosynthetic pathway.
 11. Themethod of claim 10, wherein said metabolite of said glucose pathway orsaid fatty acid biosynthetic pathway is selected from the groupconsisting of glucose, glycogen, lactate, CO₂, fatty acid, acetyl Co-A,RNA ribose and DNA deoxyribose.
 12. The method of claim 11, wherein saidmetabolite of said glucose pathway or said fatty acid biosyntheticpathway is chemically derivatized for said analysis.
 13. The method ofclaim 12, wherein said analysis comprises mass spectrometry.
 14. Themethod of claim 13, wherein said mass spectrometry is mass isotopomerdistribution analysis. 15-16. (canceled)
 17. The method of claim 1,wherein said compound of Formula Ia is a compound of formula III, or apharmaceutically acceptable salt thereof:


18. The method of claim 1, wherein said treatment is selected from thegroup consisting of oral administration, transmucosal administration,buccal administration, nasal administration, inhalation, parentaladministration, intravenous, subcutaneous, intramuscular, sublingual,transdermal administration, and rectal administration. 19-96. (canceled)97. The method of claim 1, wherein the metabolite of the compound offormula (Ia) is selected from the group consisting of

or a salt thereof, and a compound of formula MS213

wherein R₆ is selected from the group consisting of hydrogen, alkyl(C₁-C₈), alkoxy (C₁-C₈), isoquinolinones, indoles, thiazole, oxazole,oxadiazole, thiophene, or phenyl.